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Mycoplasma pneumoniae ( M. pneumoniae ) is an atypical bacterial pathogen responsible for community-acquired pneumonia primarily among school-aged children and young adults. Camellia oleifera ( C. oleifera ) has been used as a medicinal and edible plant in China for centuries, the constituents from which possessed various bioactivities. Notably, flavonoids existing in residues of C. oleifera defatted seeds exhibited significant anti-inflammatory activities. In the present study, we investigated the impact of total flavonoids from C. oleifera (TFCO) seed extract on M. pneumoniae pneumonia. TFCO was obtained using multiple column chromatography methods and identified as kaempferol glycosides via UPLC-HRESIMS. In a M. pneumoniae pneumonia mouse model, TFCO significantly reduced the lung damage, suppressed IL-1β, IL-6, and TNF-α production, and curbed TLR2 activation triggered by M. pneumoniae . Similarly, in RAW264.7 macrophage cells stimulated by lipid-associated membrane proteins (LAMPs), TFCO suppressed the generation of proinflammatory cytokines and TLR2 expression. Moreover, TFCO diminished the phosphorylation of IκBα, JNK, ERK, p38, and p65 nuclear translocation in vitro. In conclusion, TFCO alleviated M. pneumoniae -induced lung damage via inhibition of TLR2-mediated NF-κB and MAPK pathways, suggesting its potential therapeutic application in M. pneumoniae -triggered lung inflammation.

Mycoplasma pneumoniae ( M. pneumoniae , Mp) is an intracellular pathogen that causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans and can infect and survive in the host cells leading to excessive immune responses. Extracellular vesicles (EVs) from host cells carry components of pathogens to recipient cells and play a role in intercellular communication during infection. However, there is limited knowledge on whether EVs derived from M. pneumoniae -infected macrophages play as intercellular messengers and functional mechanisms. In this study, we establish a cell model of M. pneumoniae -infected macrophages that continuously secrete EVs to further asses their role as intercellular messengers and their functional mechanisms. Based on this model, we determined a method for isolating the pure EVs from M. pneumoniae -infected macrophages, which employs a sequence of operations, including differential centrifugation, filtering, and ultracentrifugation. We identified EVs and their purity using multiple methods, including electron microscopy, nanoparticle tracking analysis, Western blot, bacteria culture, and nucleic acid detection. EVs from M. pneumoniae -infected macrophages are pure, with a 30-200 nm diameter. These EVs can be taken up by uninfected macrophages and induce the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-8 through the nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPK) signals pathway. Moreover, the expression of inflammatory cytokines induced by EVs relies on TLR2-NF-κB/JNK signal pathways. These findings will help us better understand a persistent inflammatory response and cell-to-cell immune modulation in the context of M. pneumoniae infection.

Mycoplasma pneumoniae, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent. Lipidomics analysis shows that P116 specifically extracts lipids such as phosphatidylcholine, sphingomyelin and cholesterol. Structures of different conformational states reveal the mechanism by which lipids are extracted. This finding immediately suggests a way to control Mycoplasma infection by interfering with lipid uptake., (© 2023. The Author(s).)

Mycoplasma pneumoniae is the most common pathogen of respiratory tract infection in children and adults. Clinical observation shows that M . pneumoniae infection can cause massive mucus secretion in the respiratory tract, which makes the breathing of patients difficult. Studies have shown that M . pneumoniae infection can cause massive secretion of mucin 5AC (MUC5AC). Adhesin P1 plays an important role in the pathogenesis of M . pneumoniae infection by mediating the adhesion of pathogens to host cells, and the C-terminal residues of P1 (P1-C) are immunogenic. This study investigated the molecular mechanism of Wnt/β-catenin signaling pathway inhibitor Dickkopf-1 (DKK1) in the secretion of MUC5AC in mouse airway epithelial cells (MAECs) induced by P1-C. Scanning electron microscope and hematoxylin-eosin staining were used to observe the effect of P1-C on mucus secretion of MAECs. Protein chip was used to detect the secretion of cytokines and analyse the enrichment of related signaling pathways induced by P1-C in MAECs. Periodic acid schiff stain (PAS) staining, Tunel staining and Masson staining were used to detect the damage of the lungs of mouse exposed to P1-C. Immunohistochemistry was used to detect the secretion of MUC5AC expression, and Western blotting was used to reveal the molecular mechanism of DKK1-regulated secretion of MUC5AC induced by P1-C protein in MACES. The results showed that P1-C induced the massive secretion of mucus and inflammatory factors in MAECs. During P1-C infection, DKK1 down-regulated janus kinase 2 (JAK2), phosphorylation signaling and transcription activator 1 (p-STAT1) and phosphorylation signaling and activator of transcription 3 (p-STAT3) expression. Overexpression of DKK1 significantly up-regulated the expression of MUC5AC repressor transcription factor fork-head box protein A2 (FOXA2). At the same time, the expression of MUC5AC induced by P1-C was inhibited significantly. It is speculated that DKK1 can effectively reduce the secretion of MUC5AC in MAECs induced by P1-C by inhibiting the JAK/STAT1-STAT3 signaling pathway and up-regulating the expression of FOXA2.

The filamentous fungus Aspergillus oryzae , also known as koji mold, has been used for centuries in the production of fermented foods in East Asia. A. oryzae fermentation can produce enzymes and metabolites with various bioactivities. In this study, we investigated whether A. oryzae fermentation extract (AOFE) has any effect on Mycoplasma pneumoniae (Mp) pneumonia. We performed solid-state fermentation of A. oryzae and obtained the ethanol extract. AOFE was analyzed by HPLC, and the major component was identified to be kojic acid. In vitro, AOFE suppressed Mp growth and invasion into A549 lung epithelial cells as determined by the gentamicin protection assay. AOFE treatment also suppressed Mp-stimulated production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 at mRNA and protein levels in murine MH-S alveolar macrophages. In a mouse model of Mp pneumonia, Mp infection induced a marked pulmonary infiltration of neutrophils, which was significantly reduced in mice pre-treated orally with AOFE. AOFE administration also suppressed the production of proinflammatory cytokines and chemokines in the lungs. Collectively, our results show that AOFE has the potential to be developed into a preventive/therapeutic agent for Mp pneumonia.

Mycoplasma pneumoniae (M. pneumoniae) is the most common pathogen of respiratory tract infections in both children and adults. M. pneumoniae P1 adhesin plays an important role in the pathogenesis of M. pneumoniae infection by mediating the attachment of pathogen to host cells. The inoculation of C-terminal residuals of P1 (P1-C) showed a protective role from M. pneumoniae infection. Accumulated evidence suggests that the Wnt/β-Catenin signaling is implicated in regulation of inflammatory responses to bacterial infections. However, mechanisms underlying the regulatory roles of Wnt signaling in host cells in response to M. pneumoniae infections are incompletely understood. In the present study, the impact and molecular mechanism of Wnt/β-catenin signaling in immune responses induced by M. pneumoniae P1-C were investigated. The results demonstrated that the P1-C could activate Wnt/β-catenin and Toll-like receptor (TLR) signaling in primary mouse airway epithelial cells cultured in an air-liquid interface (ALI) state. Interestingly, the inhibition of Wnt/β-catenin signaling by an adenovirus-mediated Wnt inhibitor Dickkopf-1 (Dkk1) gene transduction alleviated the P1-C induced inflammation fibrosis in mouse lung, accompanied by the reduced expression of epithelial mesenchymal transition (EMT) markers. Mechanistical analysis further demonstrated that the Dkk1 could suppress the expression of JAK2/STAT1-STAT3 and Caspase3, 8/Bax signaling in mouse lung tissues. In vitro study further revealed that XAV939, a small molecule of Wnt/β-catenin inhibitor, inhibited the P1-C-activated TLR4/MyD88 signaling and cytokine productions in primary mouse airway ALI epithelial cells. This study thus provides an insight into the function of Wnt/β-catenin signaling in regulation of the pathogenesis of M. pneumoniae infection, suggesting that targeting Wnt/β-catenin signaling by gene transduction of Dkk1, or pharmacological molecules of inhibitor may be a promised approach that worthy of further investigation in the treatment of M. pneumoniae pneumonia., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mycoplasma pneumoniae is a small cell wall-lacking bacterium that is a common cause of bronchitis and pneumonia in humans. In addition to its clinical importance, M. pneumoniae has recently been considered a promising model organism for synthetic biology because of its small genome size and unique cell structure. At one cell pole, M. pneumoniae forms the attachment organelle that is responsible for adherence to host cells and gliding motility. The attachment organelle is a membrane protrusion and is composed of number of molecules, including adhesin and cytoskeletal proteins. Genetic manipulation techniques are key research approaches for understanding the structure and the function of this unique molecular machinery. In this chapter, standard genetic engineering methods for this species using the Tn4001 transposon vector are described., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Pneumonia is a serious infectious disease with increased morbidity and mortality worldwide. The M. pneumoniae is a major airway pathogen that mainly affects respiratory tract and ultimately leads to the development of pneumonia. The current exploration was aimed to uncover the beneficial properties of pinocembrin against the M. pneumoniae-triggered pneumonia in mice via its anti-inflammatory property. The pneumonia was stimulated to the BALB/c mice via infecting them with M. pneumoniae (100 µl) for 2 days through nasal drops and concomitantly treated with pinocembrin (10 mg/kg) for 3 days. The azithromycin (100 mg/kg) was used as a standard drug. Then the lung weight, nitric oxide, and myeloperoxidase (MPO) activity was assessed. The content of MDA, GSH, and SOD activity was scrutinized using kits. The total cells and DNA amount present in the bronchoalveolar lavage fluid (BALF) was assessed by standard methods. The IL-1, IL-6, IL-8, TNF-α, and TGF contents in the BALF samples and NF-κB level in the lung tissues were assessed using kits. The lung histopathology was assessed microscopically to detect the histological alterations. The 10 mg/kg of pinocembrin treatment substantially decreased the lung weight, nitric oxide (NO) level, and MPO activity. The MDA level was decreased, and GSH content and SOD activity were improved by the pinocembrin treatment. The pinocembrin administered pneumonia animals also demonstrated the decreased total cells, DNA amount, IL-1, IL-6, IL-8, TNF-α, and TGF in the BALF and NF-κB level. The findings of histological studies also witnessed the beneficial role of pinocembrin against M. pneumoniae-infected pneumonia. In conclusion, our findings confirmed that the pinocembrin effectively ameliorated the M. pneumoniae-provoked inflammation and oxidative stress in the pneumonia mice model. Hence, it could be a hopeful therapeutic agent to treat the pneumonia in the future., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells 1 . Here we use advances in cryo-electron tomography and sub-tomogram analysis 2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae. To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes 4 . By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells., (© 2022. The Author(s).)

Mycoplasma pneumoniae ( M. pneumoniae ) is the most common cause of community-acquired pneumonia. Toll-like receptors (TLRs) play an essential role in pneumonia. The purpose of this study was to investigate the roles of TLR4 in M. pneumoniae . Mice were administrated with 100  μ l (1 × 107 ccu/ml) of M. pneumoniae . HE staining was applied for histological analysis. The protein expression was determined by western blot. The cytokine level was detected by ELISA. The results showed that TLR4-deficient mice were protected from M. pneumoniae . However, downregulation of TLR4 inhibited inflammatory response and autophagy. Moreover, transcription factor EB (TFEB) participated in M. pneumoniae -induced inflammatory response and autophagy, while knockdown of TLR4 downregulated TFEB and its nuclear translocation., Competing Interests: The authors declared that they have no conflicts of interest regarding this work., (Copyright © 2022 Yan Liu et al.)

Background: Pneumonia is a serious pediatric lung injury disease caused by Mycoplasma pneumoniae (M. pneumoniae) with increasing global prevalence every year. The WHO has reported that nearly 19% of children die due to pneumonia worldwide., Objective: The present research was conducted to discover the ameliorative properties of geraniol against M. pneumoniae-provoked pneumonia in mice through the modulation of inflammatory responses., Methodology: The pneumonia was provoked in the male Swiss albino mice via infecting animals with 100 µl of M. pneumoniae for 2 days and supplemented concurrently with 20 mg/kg of geraniol for 3 days. 100 mg/kg of azithromycin was used as a standard drug. The nitric oxide (NO) level and MPO activity were measured using kits. The SOD activity, GSH, and MDA levels were studied using standard methods. The polymerase chain reaction (PCR) study was performed to examine the M. pneumoniae DNA load. The inflammatory cytokines status was assessed by assay kits. The ERK1/2, JNK1/2, and NF-κB expressions were studied by reverse-transcription (RT-PCR). The lung tissues were analyzed microscopically to investigate the histological alterations., Results: Geraniol treatment effectively reduced lung weight, NO level, and MPO activity in the pneumonia mice. The total cells and M. pneumoniae DNA load were also decreased by the geraniol. The SOD activity and GSH level were improved and MDA was decreased by the geraniol treatment. The IL-1, IL-6, IL-8, TNF-α, and TGF status were appreciably depleted by the geraniol in the pneumonia mice. Geraniol also suppressed the ERK1/2 and NF-κB expressions in the lung tissues. Histological findings also suggest the therapeutic roles of geraniol against pneumonia in mice., Conclusion: In summary, our results proved the beneficial roles of geraniol against the M. pneumoniae-provoked pneumonia. Geraniol could be a hopeful therapeutic agent to treat pneumonia in the future., (© 2022 Wiley Periodicals LLC.)

Studies have shown that club cell secretory protein (CC16) plays important protective roles in the lungs, yet its complete biological functions are unclear. We devised a translational mouse model in order to investigate the impact of early life infections, in the context of CC16 deficiency, on lung function in adult mice. CC16 sufficient (WT) and deficient (CC16 -/- ) mice were infected with Mycoplasma pneumoniae (Mp) as weanlings and assessed as adults ( e arly l ife i nfection m odel; ELIM) and compared to adult mice infected for only 3 days ( a dult i nfection m odel; AIM). CC16 -/- Mp-infected mice had significantly increased airway hyperresponsiveness (AHR) in both models compared to WT mice. However, CC16 -/- mice infected in early life (ELIM) displayed significantly increased AHR compared to CC16 -/- mice infected in adulthood (AIM). In stark contrast, lung function in ELIM WT mice returned to levels similar to saline-treated controls. While WT mice cleared Mp infection in the ELIM, CC16 -/- mice remained colonized with Mp throughout the model, which likely contributed to increased airway remodeling and persistence of Muc5ac expression. When CC16 -/- mouse tracheal epithelial cells (MTECs) were infected with Mp, increased Mp colonization and collagen gene expression were also detected compared to WT cells, suggesting that CC16 plays a protective role during Mp infection, in part through epithelial-driven host defense mechanisms.

Mycoplasma pneumoniae (M. pneumoniae) is an important pathogen in community-acquired pneumonia. The community-acquired respiratory distress syndrome (CARDS) toxin is the only known virulence factor of M. pneumoniae. It is worth exploring whether this toxin can be used as a candidate antigen for the serodiagnosis of M. pneumoniae. In this study, the full-length, N-terminal, and C-terminal regions of the CARDS toxin were expressed and purified, and serological reactions were evaluated using ELISA. A total of 184 serum samples were collected and tested using a commercialized test kit. Eighty-seven samples were positive, and 97 samples were negative for infection. The purified recombinant proteins were used as antigens to test the serum via indirect ELISA. The sensitivity of the CARDS toxin, the N-terminal region, and the C-terminal region were 90.8%, 90.8%, and 92.0%, respectively. The specificity of the CARDS toxin, the N-terminal region, and the C-terminal region were 85.6%, 73.2%, and 93.8%, respectively. All three CARDS toxin proteins exhibited good reactivity, of which the C-terminal region had a good discrimination ability in human sera. This may have a potential diagnostic value for M. pneumoniae infections., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalized children in the United States. It is also responsible for a spectrum of other respiratory tract disorders and extrapulmonary manifestations in children and adults. The main virulence factor of M. pneumoniae is a 591 amino acid multifunctional protein called Community Acquired Respiratory Distress Syndrome (CARDS) toxin. The amino terminal region of CARDS toxin (N-CARDS) retains ADP-ribosylating activity and the carboxy region (C-CARDS) contains the receptor binding and vacuolating activities. After internalization, CARDS toxin is transported in a retrograde manner from endosome through the Golgi complex into the endoplasmic reticulum. However, the mechanisms and criteria by which internalized CARDS toxin is transported and activated to execute its cytotoxic effects remain unknown. In this study, we used full-length CARDS toxin and its mutant and truncated derivatives to analyze how pharmacological drugs that alter pH of intracellular vesicles and electrical potential across vesicular membranes affect translocation of CARDS toxin in mammalian cells. Our results indicate that an acidic environment is essential for CARDS toxin retrograde transport to endoplasmic reticulum. Moreover, retrograde transport facilitates toxin clipping and is required to induce vacuole formation. Additionally, toxin-mediated cell vacuolation is strictly dependent on the function of vacuolar type-ATPase.

Mycoplasma genitalium (Mge) and Mycoplasma pneumoniae (Mpn) are two human pathogens associated with urogenital and respiratory tract infections, respectively. The recent elucidation of the tridimensional structure of their major cytoadhesins by X-ray crystallography and cryo-electron microscopy/tomography, has provided important insights regarding the mechanics of infection and evasion of immune surveillance., Competing Interests: Declaration of Interests No interests are declared., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mycoplasma pneumoniae is a significant cause of pneumonia and post infection sequelae affecting organ sites distant to the respiratory tract are common. It is also a model organism where extensive 'omics' studies have been conducted to gain insight into how minimal genome self-replicating organisms function. An N-terminome study undertaken here identified 4898 unique N-terminal peptides that mapped to 391 (56%) predicted M. pneumoniae proteins. True N-terminal sequences beginning with the initiating methionine (iMet) residue from the predicted Open Reading Frame (ORF) were identified for 163 proteins. Notably, almost half (317; 46%) of the ORFS derived from M. pneumoniae strain M129 are post-translationally modified, presumably by proteolytic processing, because dimethyl labelled neo-N-termini were characterised that mapped beyond the predicted N-terminus. An analysis of the N-terminome describes endoproteolytic processing events predominately targeting tryptic-like sites, though cleavages at negatively charged residues in P1' (D and E) with lysine or serine/alanine in P2' and P3' positions also occurred frequently. Surfaceome studies identified 160 proteins (23% of the proteome) to be exposed on the extracellular surface of M. pneumoniae. The two orthogonal methodologies used to characterise the surfaceome each identified the same 116 proteins, a 72% (116/160) overlap. Apart from lipoproteins, transporters, and adhesins, 93/160 (58%) of the surface proteins lack signal peptides and have well characterised, canonical functions in the cell. Of the 160 surface proteins identified, 134 were also targets of endo-proteolytic processing. These processing events are likely to have profound implications for how the host immune system recognises and responds to M. pneumoniae.

The overactivation of macrophages causes chronic inflammatory diseases. Short-chain fatty acids (SCFAs), potential drugs for clinical treatment, are modulators of macrophage inflammatory reaction. Therefore, the modulation of macrophage-mediated cell activity is expected to become a new therapeutic strategy for inflammatory diseases caused by Mycoplasma pneumoniae. In this study, 2 kinds of SCFAs (propionate and butyrate) were found to have anti-inflammatory effects in M. pneumoniae-stimulated THP-1 cells inflammatory. They inhibited the expressions of IL-4, IL-6, ROS, and NLRP3 inflammasome, while enhancing the expressions of IL-10 and IFN-γ. Our study revealed these 2 agents to repress transcriptional activities of NF-κB, which are important modulators of inflammation. Meanwhile, SCFAs can significantly enhance the autophagy induced by M. pneumoniae. Considering that SCFAs have few side effects, they might be the promising adjuvant therapy for the prevention and/or treatment of various inflammatory diseases., (© The Author(s) 2020. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

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.

Background: Chlamydia pneumoniae and Mycoplasma pneumoniae have been implicated in the pathogenesis of asthma and are responsible for chronic inflammation when host immune system fails to eradicate the bacteria., Method: We performed a prospective study on 410 patients who underwent a visit at the asthma clinic of CHU of Liege between June 2016 and June 2018 with serology testing for C. pneumoniae and M. pneumoniae., Results: 65% of our asthmatic population had serum IgA and/or IgG towards C. pneumoniae, while only 12.6% had IgM and/or IgG against M. pneumoniae. Compared to seronegative asthmatics, asthmatics with IgA+ and IgG+ against C. pneumoniae were more often male and older with a higher proportion of patients with smoking history. They received higher doses of inhaled corticosteroids (ICS) and displayed lower FEV 1 /FVC ratio, higher RV/TLC ratio and lower conductance. They had higher levels of fibrinogen, though in the normal range and had lower sputum eosinophil counts. Patients with IgA- and IgG+ against C. pneumoniae were older and had higher blood monocyte counts and alpha-1-antitrypsin levels as compared to seronegative patients. Patients with IgM and/or IgG towards M. pneumoniae were more often males than seronegative asthmatics. In a subpopulation of 14 neutrophilic asthmatics with Chlamydia pneumoniae IgA + /IgG + treated with macrolides, we found a significant decrease in blood neutrophils and normalization of sputum neutrophil count but no effect on asthma quality of life and exacerbations., Conclusion: Positive Chlamydia serologic test is more common than positive Mycoplasma serology. Asthmatics with IgA and IgG against C. pneumoniae have more severe disease with increased airway obstruction, higher doses of ICS, more signs of air trapping and less type-2 inflammation.

Mycoplasma pneumoniae is a slow-growing, human pathogen that causes atypical pneumonia. Because it lacks a cell wall, many antibiotics are ineffective. Due to its reduced genome and dearth of many biosynthetic pathways, this fastidious bacterium depends on rich, undefined medium for growth, which makes large-scale cultivation challenging and expensive. To understand factors limiting growth, we developed a genome-scale, constraint-based model of M. pneumoniae called iEG158_mpn to describe the metabolic potential of this bacterium. We have put special emphasis on cell membrane formation to identify key lipid components to maximize bacterial growth. We have used this knowledge to predict essential components validated with in vitro serum-free media able to sustain growth. Our findings also show that glycolysis and lipid metabolism are much less efficient under hypoxia; these findings suggest that factors other than metabolism and membrane formation alone affect the growth of M. pneumoniae. Altogether, our modelling approach allowed us to optimize medium composition, enabled growth in defined media and streamlined operational requirements, thereby providing the basis for stable, reproducible and less expensive production.

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the conducting airways, causing bronchitis and atypical or "walking" pneumonia in humans. M. pneumoniae recognizes sialylated and sulfated oligosaccharide receptors to colonize the respiratory tract, but the contribution of the latter is particularly unclear. We used chamber slides coated with sulfatide (3- O -sulfogalactosylceramide) to provide a baseline for M. pneumoniae binding and gliding motility. As expected, M. pneumoniae bound to surfaces coated with sulfatide in a manner that was dependent on sulfatide concentration and incubation temperature and inhibited by competing dextran sulfate. However, mycoplasmas bound to sulfatide exhibited no gliding motility, regardless of receptor density. M. pneumoniae also bound lactose 3'-sulfate ligated to an inert polymer scaffold, and binding was inhibited by competing dextran sulfate. The major adhesin protein P1 mediates adherence to terminal sialic acids linked α-2,3, but P1-specific antibodies that blocked M. pneumoniae hemadsorption (HA) and binding to the sialylated glycoprotein laminin by 95% failed to inhibit mycoplasma binding to sulfatide, suggesting that P1 does not mediate binding to sulfated galactose. Consistent with this conclusion, the M. pneumoniae HA-negative mutant II-3 failed to bind to sialylated receptors but adhered to sulfatide in a temperature-dependent manner., (Copyright © 2020 American Society for Microbiology.)

Interleukin-1β (IL-1β) plays pivotal roles in controlling bacterial infections and is produced after the processing of pro-IL-1β by caspase-1, which is activated by the inflammasome. In addition, caspase-1 cleaves the cytosolic protein, gasdermin-D (GSDMD), whose N-terminal fragment subsequently forms a pore in the plasma membrane, leading to the pyroptic cell-death-mediated release of IL-1β. Living cells can also release IL-1β via GSDMD pores or other unconventional secretory pathways. However, the precise mechanisms are poorly defined. Here, we show that lipoproteins from Mycoplasma salivarium (MsLP) and Mycoplasma pneumoniae (MpLP) and an M. salivarium-derived lipopeptide (FSL-1), which are activators of the nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, induce IL-1β release from mouse bone-marrow-derived macrophages (BMMs) without inducing cell death. The levels of IL-1β release induced by MsLP, MpLP and FSL-1 were more than 100 times lower than those induced by the canonical NLRP3 activator nigericin. The IL-1β release-inducing activities of MsLP, MpLP and FSL-1 were not attenuated in BMMs from GSDMD-deficient mice. Furthermore, both active caspase-1 and cleaved GSDMD were detected in response to transfection of FSL-1 into the cytosol of BMMs, but the release of IL-1β was unaffected by GSDMD deficiency. Meanwhile, punicalagin, a membrane-stabilizing agent, drastically down-regulated the release of IL-1β in response to FSL-1. These results suggest that mycoplasmal lipoprotein/lipopeptide-induced IL-1β release by living macrophages is not mediated via GSDMD but rather through changes in membrane permeability., (© 2020 John Wiley & Sons Ltd.)

Structural biology studies performed inside cells can capture molecular machines in action within their native context. In this work, we developed an integrative in-cell structural approach using the genome-reduced human pathogen Mycoplasma pneumoniae We combined whole-cell cross-linking mass spectrometry, cellular cryo-electron tomography, and integrative modeling to determine an in-cell architecture of a transcribing and translating expressome at subnanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome, and the transcription elongation factors NusG and NusA. We pinpointed NusA at the interface between a NusG-bound elongating RNAP and the ribosome and propose that it can mediate transcription-translation coupling. Translation inhibition dissociated the expressome, whereas transcription inhibition stalled and rearranged it. Thus, the active expressome architecture requires both translation and transcription elongation within the cell., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Here, we propose an approach to identify active metabolic pathways by integrating gene essentiality analysis and protein abundance. We use two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene content similarity yet show significant metabolic differences. First, we build detailed metabolic maps of their carbon metabolism, the most striking difference being the absence of two key enzymes for glucose metabolism in M. agalactiae. We then determine carbon sources that allow growth in M. agalactiae, and we introduce glucose-dependent growth to show the functionality of its remaining glycolytic enzymes. By analyzing gene essentiality and performing quantitative proteomics, we can predict the active metabolic pathways connected to carbon metabolism and show significant differences in use and direction of key pathways despite sharing the large majority of genes. Gene essentiality combined with quantitative proteomics and metabolic maps can be used to determine activity and directionality of metabolic pathways., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study was designed to investigate the effect of exogenous hydrogen sulfide (H 2 S) on the secretion of Heme oxygenase (HO-1) and proinflammatory cytokines in human mononuclear cell line THP-1 stimulated by lipid-associated membrane proteins (LAMPs) prepared from Mycoplasma pneumoniae (M. pneumoniae) and explore its regulatory mechanism. Cultured cells were stimulated with M. pneumoniae LAMPs after pretreatment with H 2 S to analyze the production of proinflammatory cytokines and HO-1 by enzyme-linked immunosorbent assay (ELISA) and Western blot. The results showed that THP-1 cells, which were stimulated by LAMPs after pretreatment with H 2 S, had decreased production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by inhibiting the mitogen-activated protein kinases (MAPKs)/nuclear factor-kappa B (NF-κB) signaling pathway and increased expression of HO-1 by activating the nuclear factor E2-related factor 2 (Nrf2) signaling pathway. Our results indicate that H 2 S may play an important role in attenuating inflammation induced by M. pneumoniae LAMPs due to its ability to decrease the production of IL-6 and IL-8 and increase the expression of the HO-1. These findings support further studies for possible clinical applications.

The C-terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C-terminal sequence and levels of protein expression remains unknown. Here, we identified C-terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C-terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The aim of this study was to evaluate the changes in the three subsets of monocyte (classical, intermediate, and non-classical) and the expression of human leukocyte antigen-DR (HLA-DR) on monocyte subsets during MP pneumonia in children. Monocyte subsets were analyzed in the peripheral blood of healthy volunteers and MP pneumonia patients at the stages of admission and remission after clinical therapy. They were defined as classical (CD14 + CD16 - ), intermediate (CD14 bright CD16 + ), and non-classical (CD14 dim CD16 + ) using flow cytometry. Furthermore, three subsets of monocyte were analyzed for the expression of HLA-DR. Patients with MP pneumonia at admission had a higher proportion of intermediate and non-classical monocytes than healthy subjects (all P < 0.05). The proportion of intermediate subset and non-classical subset was lower in MP pneumonia patients at remission than at admission (all P < 0.05). In comparison with the other monocyte subsets, intermediate subset showed a significantly higher percentage of HLA-DR in MP pneumonia patients at admission (P < 0.05). Further analysis revealed that the expression of HLA-DR on intermediate subset was lower in severe patients than in non-severe patients (P < 0.05).Our data has shown for the first time that MP pneumonia is associated with the increased proportion of non-classical and intermediate monocytes, indicating the involvement of monocyte-related mechanisms in the pathogenesis of this disease. Additionally, the decreased expression of HLA-DR on CD14 bright CD16 + subset may be a potential indicator of the severity of MP pneumonia.

Background Discriminating Mycoplasma pneumoniae (MP) from Streptococcus pneumoniae (SP) and viral etiologies of community-acquired pneumonia (CAP) is challenging but has important implications regarding empiric antibiotic therapy. We investigated patient parameters upon hospital admission to predict MP infection. Methods All patients hospitalized in a tertiary care hospital between 2013 and 2017 for CAP with a confirmed etiology were analyzed using logistic regression analyses and area under the receiver operator characteristics (ROC) curves (AUC) for associations between demographic, clinical and laboratory features and the causative pathogen. Results We analyzed 568 patients with CAP, including 47 (8%) with MP; 152 (27%) with SP and 369 (65%) with influenza or other viruses. Comparing MP and SP by multivariate logistic regression analysis, younger age (odds ration [OR] 0.56 per 10 years, 95% CI 0.42-0.73), a lower neutrophil/lymphocyte ratio (OR 0.9, 0.82-0.99) and an elevated C-reactive protein/procalcitonin (CRP/PCT) ratio (OR 15.04 [5.23-43.26] for a 400 mg/μg cut-off) independently predicted MP. With a ROC curve AUC of 0.91 (0.80 for the >400 mg/μg cutoff), the CRP/PCT ratio was the strongest predictor of MP vs. SP. The discriminatory value resulted from significantly lower PCT values (p < 0.001) for MP, while CRP was high in both groups (p = 0.057). Comparing MP and viral infections showed similar results with again the CRP/PCT ratio providing the best information (AUC 0.83; OR 5.55 for the >400 mg/μg cutoff, 2.26-13.64). Conclusions In patients hospitalized with CAP, a high admission CRP/PCT ratio predicts M. pneumoniae infection and may improve empiric management.

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia among hospitalised children in United States and worldwide. Community-acquired respiratory distress syndrome (CARDS) toxin is a key virulence determinant of M. pneumoniae. The N-terminus of CARDS toxin exhibits ADP-ribosyltransferase (ADPRT) activity, and the C-terminus possesses binding and vacuolating activities. Thiol-trapping experiments of wild-type (WT) and cysteine-to-serine-mutated CARDS toxins with alkylating agents identified disulfide bond formation at the amino terminal cysteine residues C230 and C247. Compared with WT and other mutant toxins, C247S was unstable and unusable for comparative studies. Although there were no significant variations in binding, entry, and retrograde trafficking patterns of WT and mutated toxins, C230S did not elicit vacuole formation in intoxicated cells. In addition, the ADPRT domain of C230S was more sensitive to all tested proteases when compared with WT toxin. Despite its in vitro ADPRT activity, the reduction of C230S CARDS toxin-mediated ADPRT activity-associated IL-1β production in U937 cells and the recovery of vacuolating activity in the protease-released carboxy region of C230S indicated that the disulfide bond was essential not only to maintain the conformational stability of CARDS toxin but also to properly execute its cytopathic effects., (© 2019 John Wiley & Sons Ltd.)

Epithelial cell shedding is a defence mechanism against infectious microbes that use these cells as an infection foothold and that eliminate microbes from infection foci by removing infected cells. Mycoplasma pneumoniae, a causative agent of respiratory infections, is known to adhere to and colonise the surface of ciliated airway epithelial cells; it produces a large amount of hydrogen peroxide, indicating its capability of regulating hydrogen peroxide-induced infected cell detachment. In this study, we found that M. pneumoniae reduces exogenous hydrogen peroxide-induced detachment of the infected cells from culture plates. This cell detachment occurred dependently of DNA damage-initiated, poly (ADP-ribose) polymerase 1 (PARP1)-mediated cell death, or parthanatos. In cells infected with M. pneumoniae, exogenous hydrogen peroxide failed to induce DNA damage-initiated poly (ADP-ribose) (PAR) synthesis and concomitant increased cytoplasmic membrane rupture, both of which are biochemical hallmarks of parthanatos. The impairment of PAR synthesis was attributed to a reduction in the amount of cytosolic nicotinamide adenine dinucleotide (NAD), a substrate of PARP1, caused by M. pneumoniae. On the other hand, nonadherent mutant strains of M. pneumoniae showed a lower ability to reduce cell detachment than wild-type strains, but the extent to which NAD was decreased in infected cells was comparable to that seen in the wild-type strain. We found that NAD depletion could induce PARP1-independent cell detachment pathways following stimulation with hydrogen peroxide and that M. pneumoniae could also regulate PARP1-independent cell detachment in a cytoadhesion-dependent manner. These results suggest that M. pneumoniae might regulate infected cell detachment induced by hydrogen peroxide that it produces itself, and such a mechanism may contribute to sustaining the bacterial infection., (© 2019 John Wiley & Sons Ltd.)

Excessive inflammation is fundamental in the pathophysiology of Mycoplasma pneumoniae (MP)-induced respiratory infection in children. Histone deacetylase 5 (HDAC5) is involved in the regulation of inflammation, however, whether it associates with immunity against MP infection is not determined. We report here that HDAC5 expression is decreased in peripheral blood mononuclear cells (PBMCs) from Mycoplasma pneumoniae pneumonia (MPP) children as well as in MP-infected peritoneal and THP-1 macrophages. Functionally, HDAC5 overexpression promotes and its depletion inhibits MP-induced proinflammatory cytokine production in THP-1 macrophages. Mechanistically, HDAC5 modulates NF-κB activation in MP-infected THP-1 macrophages, and moreover, inhibition of NF-κB activity via pharmacological inhibitor Bay 11-7082 attenuates the promotive effect of HDAC5 on MP-induced proinflammatory cytokine production in THP-1 macrophages, hence suggesting that HDAC5 promotes MP-induced inflammatory response in macrophages through NF-κB activation. Together, this study reveals a novel function of HDAC5 in promoting MP-induced inflammation and implies the possible clinical significance in controlling inflammation that underlies MMP pathophysiology., (Copyright © 2019. Published by Elsevier Inc.)

Mycoplasma pneumoniae pneumonia (MPP) is the most common respiratory infection in young children and its incidence has increased worldwide. In this study, high expression of chemokine ligand 5 (CCL5) was observed in the serum of MPP patients, and its expression was positively correlated to DNA of M. pneumoniae (MP-DNA). In vitro, M. pneumoniae (MP) infection to A549 cells induced the expression of CCL5, chemokines receptor 4 (CCR4), nuclear factor-κB (NF-κB) nuclear protein, and phosphorylation of NF-κB-p65 (p-NF-κB-p65), whereas NF-κB cytoplasmic protein was decreased. On the contrary, treatment of hyperoside counteracted the induction of MP infection and promoted the proliferation of MP-infected A549 cells. Similarly, MP-induced IL-8 and TNF-α production was also markedly reduced by hyperoside. And CCR4 inhibitor AZD2098 had a better effect than hyperoside. In addition, CCL5 recombinant protein inhibited the effect of hyperoside to promote IL-8 and TNF-α production and CCR4 expression. These results indicated that CCL5 may be involved in the progression of MPP, and hyperoside was beneficial for MPP probably through CCL5-CCR4 interactions, which may provide a potential effective therapy for MPP.

Bacteria of the genus Mycoplasma have recently attracted considerable interest as model organisms in synthetic and systems biology. In particular, Mycoplasma pneumoniae is one of the most intensively studied organisms in the field of systems biology. However, the genetic manipulation of these bacteria is often difficult due to the lack of efficient genetic systems and some intrinsic peculiarities such as an aberrant genetic code. One major disadvantage in working with M. pneumoniae is the lack of replicating plasmids that can be used for the complementation of mutants and the expression of proteins. In this study, we have analysed the genomic region around the gene encoding the replication initiation protein, DnaA, and detected putative binding sites for DnaA (DnaA boxes) that are, however, less conserved than in other bacteria. The construction of several plasmids encompassing this region allowed the selection of plasmid pGP2756 that is stably inherited and that can be used for genetic experiments, as shown by the complementation assays with the glpQ gene encoding the glycerophosphoryl diester phosphodiesterase. Plasmid-borne complementation of the glpQ mutant restored the formation of hydrogen peroxide when bacteria were cultivated in the presence of glycerol phosphocholine. Interestingly, the replicating plasmid can also be used in the close relative, Mycoplasma genitalium but not in more distantly related members of the genus Mycoplasma. Thus, plasmid pGP2756 is a valuable tool for the genetic analysis of M. pneumoniae and M. genitalium.

Mycoplasma pneumoniae is a common cause of human respiratory tract infections, including bronchitis and atypical pneumonia. M. pneumoniae binds glycoprotein receptors having terminal sialic acid residues via the P1 adhesin protein. Here, we explored the impact of sialic acid presentation on M. pneumoniae adherence and gliding on surfaces coated with sialylated glycoproteins, or chemically functionalized with α-2,3- and α-2,6-sialyllactose ligated individually or in combination to a polymer scaffold in precisely controlled densities. In both models, gliding required a higher receptor density threshold than adherence, and receptor density influenced gliding frequency but not gliding speed. However, very high densities of α-2,3-sialyllactose actually reduced gliding frequency over peak levels observed at lower densities. Both α-2,3- and α-2,6-sialyllactose supported M. pneumoniae adherence, but gliding was only observed on the former. Finally, gliding on α-2,3-sialyllactose was inhibited on surfaces also conjugated with α-2,6-sialyllactose, suggesting that both moieties bind P1 despite the inability of the latter to support gliding. Our results indicate that the nature and density of host receptor moieties profoundly influences M. pneumoniae gliding, which could affect pathogenesis and infection outcome. Furthermore, precise functionalization of polymer scaffolds shows great promise for further analysis of sialic acid presentation and M. pneumoniae adherence and gliding., (© 2018 John Wiley & Sons Ltd.)

Mycoplasma pneumoniae expresses β-glycolipids (β-GGLs) in cytoplasmic membranes, which possess a unique β(1 → 6)-linked disaccharide epitope, which has high potential in biochemical and medicinal applications. In the present study, a series of β-GGLs homologues with different acyl chains (C12, C14, C16, and C18) were prepared from a common precursor. An ELISA assay using an anti-(β-GGLs) monoclonal antibody indicated that the synthetic homologues with long acyl chains had greater diagnostic potential in the order C18 > C16 > C14 > C12. Toward a simultaneous detection of natural glycolipids by mass spectrometry (MS), a deuterium-labeled C16 homologue (β-GGL-C16-d 3 ) was prepared and applied as an internal standard for a high-resolution electrospray ionization MS (ESI-MS) analysis. The ESI-MS analysis was used to identify and quantify acyl homologues (C16/C16, C16/C18, and C18/C18) of β-GGL-C16 in cultured M. pneumoniae. A β-GGLs homologue with a 1,2-diacetyl group (C2) was also prepared as a "water soluble" glycolipid homologue and characterized by 1 H NMR spectroscopy. We envisage that each of these chemosynthetic homologues will provide promising approaches to solve medical and biological problems associated with mycoplasma infectious diseases (MIDs)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Mycoplasma pneumoniae is an atypical bacterium that causes respiratory illnesses in humans, including pharyngitis, tracheobronchitis, and community-acquired pneumonia (CAP). It has also been directly linked to reactive airway disease, asthma, and extrapulmonary pathologies. During its colonization, M. pneumoniae expresses a unique ADP-ribosylating and vacuolating cytotoxin designated c ommunity- a cquired r espiratory d istress s yndrome (CARDS) toxin. CARDS toxin persists and localizes in the airway in CAP patients, asthmatics, and trauma patients with ventilator-associated pneumonia. Although CARDS toxin binds to specific cellular receptors, is internalized, and induces hyperinflammation, histopathology, mucus hyperplasia, and other airway injury, the intracellular trafficking of CARDS toxin remains unclear. Here, we show that CARDS toxin translocates through early and late endosomes and the Golgi complex and concentrates at the perinuclear region to reach the endoplasmic reticulum (ER). Using ER-targeted SNAP-tag, we confirmed the association of CARDS toxin with the ER and determined that CARDS toxin follows the retrograde pathway. In addition, we identified a novel CARDS toxin amino acid fingerprint, KELED, that is required for toxin transport to the ER and subsequent toxin-mediated cytotoxicity. IMPORTANCE Mycoplasma pneumoniae , a leading cause of bacterial community-acquired pneumonia (CAP) among children and adults in the United States, synthesizes a 591-amino-acid ADP-ribosylating and vacuolating protein, designated c ommunity- a cquired r espiratory d istress s yndrome (CARDS) toxin. CARDS toxin alone is sufficient to induce and mimic major inflammatory and histopathological phenotypes associated with M. pneumoniae infection in rodents and primates. In order to elicit its ADP-ribosylating and vacuolating activities, CARDS toxin must bind to host cell receptors, be internalized via clathrin-mediated pathways, and subsequently be transported to specific intracellular organelles. Here, we demonstrate how CARDS toxin utilizes its unique KELED sequence to exploit the retrograde pathway machinery to reach the endoplasmic reticulum (ER) and fulfill its cytopathic potential. The knowledge generated from these studies may provide important clues to understand the mode of action of CARDS toxin and develop interventions that reduce or eliminate M. pneumoniae -associated airway and extrapulmonary pathologies., (Copyright © 2018 Ramasamy et al.)

Mycoplasma pneumoniae is a common cause of community-acquired infections of the human respiratory tract. The strongly reduced genome of the cell wall-less bacteria results in limited metabolic pathways and a small number of known virulence factors. In addition to the well-characterized adhesion apparatus and the expression of tissue-damaging substances, surface-exposed proteins with a primary function in cytosol-located processes such as glycolysis have been attracting attention in recent years. Due to interactions with host factors, it has been suggested that these bacterial proteins contribute to pathogenesis. Here, we investigated the chaperones GroEL and DnaK of M. pneumoniae as candidates for such moonlighting proteins. After successful expression in Escherichia coli and production of polyclonal antisera, the localization of both chaperones on the surface of bacteria was confirmed. Binding of recombinant GroEL and DnaK to human A549 cells, to plasminogen as well as to vitronectin, fibronectin, fibrinogen, lactoferrin and laminin was demonstrated. In the presence of both recombinant proteins and host activators, plasminogen can be activated to the protease plasmin, which is able to degrade vitronectin and fibrinogen. The results of the study extend the spectrum of surface-exposed proteins in M. pneumoniae and indicate an additional role of both chaperones in infection processes., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mycoplasma pneumoniae is strongly associated with new onset asthma and asthma exacerbations. Until recently, the molecular mechanisms utilized by M. pneumoniae to influence asthma symptoms were unknown. However, we recently reported that an ADP-ribosylating and vacuolating toxin called the Community Acquired Respiratory Distress Syndrome toxin, CARDS toxin, produced by M. pneumoniae was sufficient to promote allergic inflammation and asthma-like disease in mice. A mouse model of CARDS toxin exposure was used to evaluate total and CARDS-toxin specific serum IgE responses. Mast cell sensitization, challenge, and degranulation studies determined functionality of the CARDS toxin-specific IgE. In the current study, we report that a single mucosal exposure to CARDS toxin was sufficient to increase total serum IgE and CARDS toxin-specific IgE in mice. Mice given a second mucosal challenge of CARDS toxin responded with significant increases in total and CARDS toxin-specific IgE. CARDS toxin-specific IgE bound to an N-terminal peptide of CARDS toxin but not the C-terminal peptide. Likewise, full-length CARDS toxin and the N-terminal peptide induced mast cell degranulation. Altogether, these data demonstrate that exposure to CARDS toxin is sufficient to generate functional IgE in mice. M. pneumoniae and CARDS toxin are strongly associated with asthma exacerbations raising the possibility that the CARDS toxin-specific IgE-mast cell axis contributes to disease pathogenesis.

Unlabelled: Mycoplasma pneumoniae is a human pathogen that glides on host cell surfaces with repeated catch and release of sialylated oligosaccharides. At a pole, this organism forms a protrusion called the attachment organelle, which is composed of surface structures, including P1 adhesin and the internal core structure. The core structure can be divided into three parts, the terminal button, paired plates, and bowl complex, aligned in that order from the front end of the protrusion. To elucidate the gliding mechanism, we focused on MPN387, a component protein of the bowl complex which is essential for gliding but dispensable for cytadherence. The predicted amino acid sequence showed that the protein features a coiled-coil region spanning residue 72 to residue 290 of the total of 358 amino acids in the protein. Recombinant MPN387 proteins were isolated with and without an enhanced yellow fluorescent protein (EYFP) fusion tag and analyzed by gel filtration chromatography, circular dichroism spectroscopy, analytical ultracentrifugation, partial proteolysis, and rotary-shadowing electron microscopy. The results showed that MPN387 is a dumbbell-shaped homodimer that is about 42.7 nm in length and 9.1 nm in diameter and includes a 24.5-nm-long central parallel coiled-coil part. The molecular image was superimposed onto the electron micrograph based on the localizing position mapped by fluorescent protein tagging. A proposed role of this protein in the gliding mechanism is discussed., Importance: Human mycoplasma pneumonia is caused by a pathogenic bacterium, Mycoplasma pneumoniae This tiny, 2-μm-long bacterium is suggested to infect humans by gliding on the surface of the trachea through binding to sialylated oligosaccharides. The mechanism underlying mycoplasma "gliding motility" is not related to any other well-studied motility systems, such as bacterial flagella and eukaryotic motor proteins. Here, we isolated and analyzed the structure of a key protein which is directly involved in the gliding mechanism., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Mycoplasma pneumoniae is a human pathogen causing atypical pneumonia with a minimalized and highly streamlined genome. So far, hydrogen peroxide production, cytadherence, and the ADP-ribosylating CARDS toxin have been identified as pathogenicity determinants. We have studied haemolysis caused by M. pneumoniae, and discovered that hydrogen peroxide is responsible for the oxidation of heme, but not for lysis of erythrocytes. This feature could be attributed to hydrogen sulfide, a compound that has previously not been identified as virulence factor in lung pathogens. Indeed, we observed hydrogen sulfide production by M. pneumoniae. The search for a hydrogen sulfide-producing enzyme identified HapE, a protein with similarity to cysteine desulfurases. In contrast to typical cysteine desulfurases, HapE is a bifunctional enzyme: it has both the cysteine desulfurase activity to produce alanine and the cysteine desulfhydrase activity to produce pyruvate and hydrogen sulfide. Experiments with purified HapE showed that the enzymatic activity of the protein is responsible for haemolysis, demonstrating that HapE is a novel potential virulence factor of M. pneumoniae., (© 2015 John Wiley & Sons Ltd.)

We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated '-omics' data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Mycoplasma pneumoniae type 2a strain 309 is a simplest known bacterium and is the primary cause of community acquired pneumonia in the children. It mainly causes severe atypical pneumonia as well as several other non-pulmonary manifestations such as neurological, hepatic, hemolytic anemia, cardiac diseases and polyarthritis. The size of M. pneumoniae genome (Accession number: NC&#95;016807.1) is relatively smaller as compared to other bacteria and contains 707 functional proteins, in which 204 are classified as hypothetical proteins (HPs) because of the unavailability of experimentally validated functions. The functions of the HPs were predicted by integrating a variety of protein classification systems, motif discovery tools as well as methods that are based on characteristic features obtained from the protein sequence and metabolic pathways. The probable functions of 83HPs were predicted successfully. The accuracy of the diverse tools used in the adopted pipeline was evaluated on the basis of statistical techniques of Receiver Operating Characteristic (ROC), which indicated the reliability of the functional predictions. Furthermore, the virulent HPs present in the set of 83 functionally annotated proteins were predicted by using the Bioinformatics tools and the conformational behaviours of the proteins with highest virulence scores were studied by using the molecular dynamics (MD) simulations. This study will facilitate in the better understanding of various drug resistance and pathogenesis mechanisms present in the M. pneumoniae and can be utilized in designing of better therapeutic agents., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Mycoplasma pneumoniae is an extracellular pathogen that colonizes mucosal surfaces of the respiratory tract and is associated with asthma exacerbations. Previous reports demonstrate that surfactant protein-A (SP-A) binds live M. pneumoniae and mycoplasma membrane fractions (MMF) with high affinity. Humans express a repertoire of single-amino acid genetic variants of SP-A that may be associated with lung disease, and our findings demonstrate that allelic differences in SP-A2 (Gln223Lys) affect the binding to MMF. We show that SP-A(-/-) mice are more susceptible to MMF exposure and have significant increases in mucin production and neutrophil recruitment. Novel humanized SP-A2-transgenic mice harboring the hSP-A2 223K allele exhibit reduced neutrophil influx and mucin production in the lungs when challenged with MMF compared with SP-A(-/-) mice. Conversely, mice expressing hSP-A2 223Q have increased neutrophil influx and mucin production that are similar to SP-A(-/-) mice. Using tracheal epithelial cell cultures, we show that enhanced mucin production to MMF occurs in the absence of SP-A and is not dependent upon neutrophil recruitment. Increased phosphorylation of the epidermal growth factor receptor (EGFR) was evident in the lungs of MMF-challenged mice when SP-A was absent. Pharmacologic inhibition of EGFR prior to MMF challenge dramatically reduced mucin production in SP-A(-/-) mice. These findings suggest a protective role for SP-A in limiting MMF-stimulated mucin production that occurs through interference with EGFR-mediated signaling. SP-A interaction with the EGFR signaling pathway appears to occur in an allele-specific manner that may have important implications for SP-A polymorphisms in human diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen.

Mycoplasma pneumoniae (Mp) infections cause tracheobronchitis and "walking" pneumonia, and are linked to asthma and other reactive airway diseases. As part of the infectious process, the bacterium expresses a 591-aa virulence factor with both mono-ADP ribosyltransferase (mART) and vacuolating activities known as Community-Acquired Respiratory Distress Syndrome Toxin (CARDS TX). CARDS TX binds to human surfactant protein A and annexin A2 on airway epithelial cells and is internalized, leading to a range of pathogenetic events. Here we present the structure of CARDS TX, a triangular molecule in which N-terminal mART and C-terminal tandem β-trefoil domains associate to form an overall architecture distinct from other well-recognized ADP-ribosylating bacterial toxins. We demonstrate that CARDS TX binds phosphatidylcholine and sphingomyelin specifically over other membrane lipids, and that cell surface binding and internalization activities are housed within the C-terminal β-trefoil domain. The results enhance our understanding of Mp pathogenicity and suggest a novel avenue for the development of therapies to treat Mp-associated asthma and other acute and chronic airway diseases.

Platycodin D, extract from the root of Platycodon grandiflorum, is one of the most important monomers of the Qinbaiqingfei pellets (Qinbai) that has already been approved as the first Traditional Chinese Medicine for clinic use as an anti-M. pneumoniae agent. Qinbai constituents Scutellaria baicalensis and Platycodon grandiflorum were used to treat thousands of patients clinically in China each year. In this study, a M. pneumoniae-infected mouse strain, BALB/c, and a human-derived epithelial cell line, A549 type II pneumocytes, were used as experimental model. Anti-M. pneumoniae effect of Platycodin D was measured by the Real-time quantitative PCR, while the cell pathological change with hematoxylin and eosin and the growth recovery effects were determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Trypan Blue dye in the experimental model after M. pneumoniae infection. Our research results showed that Platycodin D could significantly inhibit M. pneumoniae and promote cell growth after anti- M. pneumoniae treatment in the infected cells or mice.

MyMpn (http://mympn.crg.eu) is an online resource devoted to studying the human pathogen Mycoplasma pneumoniae, a minimal bacterium causing lower respiratory tract infections. Due to its small size, its ability to grow in vitro, and the amount of data produced over the past decades, M. pneumoniae is an interesting model organisms for the development of systems biology approaches for unicellular organisms. Our database hosts a wealth of omics-scale datasets generated by hundreds of experimental and computational analyses. These include data obtained from gene expression profiling experiments, gene essentiality studies, protein abundance profiling, protein complex analysis, metabolic reactions and network modeling, cell growth experiments, comparative genomics and 3D tomography. In addition, the intuitive web interface provides access to several visualization and analysis tools as well as to different data search options. The availability and--even more relevant--the accessibility of properly structured and organized data are of up-most importance when aiming to understand the biology of an organism on a global scale. Therefore, MyMpn constitutes a unique and valuable new resource for the large systems biology and microbiology community., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Unlabelled: Mycoplasma pneumoniae synthesizes a novel human surfactant protein A (SP-A)-binding cytotoxin, designated community-acquired respiratory distress syndrome (CARDS) toxin, that exhibits ADP-ribosylating and vacuolating activities in mammalian cells and is directly linked to a range of acute and chronic airway diseases, including asthma. In our attempt to detect additional CARDS toxin-binding proteins, we subjected the membrane fraction of human A549 airway cells to affinity chromatography using recombinant CARDS toxin as bait. A 36-kDa A549 cell membrane protein bound to CARDS toxin and was identified by time of flight (TOF) mass spectroscopy as annexin A2 (AnxA2) and verified by immunoblotting with anti-AnxA2 monoclonal antibody. Dose-dependent binding of CARDS toxin to recombinant AnxA2 reinforced the specificity of the interaction, and further studies revealed that the carboxy terminus of CARDS toxin mediated binding to AnxA2. In addition, pretreatment of viable A549 cells with anti-AnxA2 monoclonal antibody or AnxA2 small interfering RNA (siRNA) reduced toxin binding and internalization. Immunofluorescence analysis of CARDS toxin-treated A549 cells demonstrated the colocalization of CARDS toxin with cell surface-associated AnxA2 upon initial binding and with intracellular AnxA2 following toxin internalization. HepG2 cells, which express low levels of AnxA2, were transfected with a plasmid expressing AnxA2 protein, resulting in enhanced binding of CARDS toxin and increased vacuolization. In addition, NCI-H441 cells, which express both AnxA2 and SP-A, upon AnxA2 siRNA transfection, showed decreased binding and subsequent vacuolization. These results indicate that CARDS toxin recognizes AnxA2 as a functional receptor, leading to CARDS toxin-induced changes in mammalian cells., Importance: Host cell susceptibility to bacterial toxins is usually determined by the presence and abundance of appropriate receptors, which provides a molecular basis for toxin target cell specificities. To perform its ADP-ribosylating and vacuolating activities, community-acquired respiratory distress syndrome (CARDS) toxin must bind to host cell surfaces via receptor-mediated events in order to be internalized and trafficked effectively. Earlier, we reported the binding of CARDS toxin to surfactant protein A (SP-A), and here we show how CARDS toxin uses an alternative receptor to execute its pathogenic properties. CARDS toxin binds selectively to annexin A2 (AnxA2), which exists both on the cell surface and intracellularly. Since AnxA2 regulates membrane dynamics at early stages of endocytosis and trafficking, it serves as a distinct receptor for CARDS toxin binding and internalization and enhances CARDS toxin-induced vacuolization in mammalian cells., (Copyright © 2014 Somarajan et al.)