MUDr. Dana Maňasková


Vyhledávání na medicinman.cz
 

Patofyziologie mykoplasma pneumonia

Genetika

Mycoplasma genitalium

  • Nejkratší genom ze všech známých volně žijících bakterií [1]
  • S největší pravděpodobností se vyvinula z grampozitivních bakterií [1]
  • Low G+C content [5]

Mycoplasma pneumoniae

  • Unusual nutritional needs
  • Arginine nonfermenting species [5]
  • Sequence composition of 16s rRNA [5]
    • All mycoplasmas of the pneumoniae group similar 16s rRNA variations unique to the group
    • M. pneumoniae has a 6.3% variation in the conserved regions
      • V.s. due to degenerative evolution from the gram-positive eubacterial group that includes bacilli, streptococci, and lactobacilli [5]
  • Sequencing of the M. pneumoniae genome in 1996
    • 816,394 bp in size
      • Genome size of (580-2200 Kbp) [7]
    • 687 genes
    • Cca 56.6% code for essential metabolic enzymes
      • Glycolysis
      • Organic acid fermentation [5]
  • Very susceptible to loss of enzymatic function by gene mutations
    • The only buffering systems against functional loss by point mutations are for:
      • Maintenance of the pentose phosphate pathway [5]
      • Nucleotide metabolism [5]
  • Loss of function in other pathways
    • Suggested to be compensated by host cell metabolism [5]
  • Reduced genome of M. pneumoniae outright lacks a number of pathways
    • TCA cycle
    • Respiratory electron transport chain
    • Biosynthesis pathways for
      • Amino acids
      • Fatty acids
      • cholesterol
      • Purines
      • Pyrimidines [5]
  • Acquire essential building blocks from their host or the environment
  • Large portion of energy metabolism is exerted to:
    • Maintain proton gradients (up to 80%)
      • Due to the high surface area to volume ratio of M. pneumoniae cells
    • 12 – 29% of energy metabolism
      • Cell growth
        • Unusually low for bacterial cells
          • Adaptation of its parasitic lifestyle [5]
  • Uses the codon UGA to code for tryptophan rather than using it as a stop codon [5]

ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae

  • Virulence determinant
  • Virulence factor (MPN372)
  • Possibly responsible for airway cellular damage and other sequelae associated with M. pneumoniae
  • M. pneumoniae MPN372 encodes a 68-kDa protein with ADP-ribosyltransferase (ART) activity
  • Its N terminus, MPN372 contains key amino acids associated with
    • NAD binding
    • ADP-ribosylating activity
  • Similar to pertussis toxin (PTX) S1 subunit (PTX-S1)
  • MPN372 ADP ribosylates both identical and distinct mammalian proteins when compared with PTX-S1.
  • MPN372 elicits extensive vacuolization and ultimate cell death of mammalian cells
    • Progressive patterns of cytopathology in tracheal rings in organ culture
  • Dramatic seroconversion to MPN372 in patients diagnosed with M. pneumoniae-associated pneumonia
    • Indicating that this toxin is synthesized in vivo
    • Possesses highly immunogenic epitopes
  • April 25, 2006 103 (17) 6724-6729 https://doi.org/10.1073/pnas.0510644103

Adhesin proteins P1 and P40/P90 inhibice

  • Adhesin proteins P1 and P40/P90 forming a trans-membrane complex
    • That binds to sialylated oligosaccharides cell receptors
  • Cryo-EM structure from P1
    • Bound to the Fab fragment of monoclonal antibody (P1/MCA4)
    • Which stops gliding
    • Induces detachment of motile M. pneumoniae cells
  • Polyclonal antibodies generated against the N-domain of P1 or against the whole ectodomain of P40/P90
    • Have little or no effects on adhesion or motility
  • Epitope of P1MCA4, centred on loop Thr1426-Asp1438 in the small C-terminal domain of P1
    • Is inaccessible to antibodies in the “open” conformation of the adhesion complex, when ready for attachment to sialylated oligosaccharides.
  • Mutations in the highly conserved Engelman motifs
    • Found in the transmembrane helix of P40/P90,
    • Also alter adhesion and motility
  • C-terminal domain of P1
    • Large conformational rearrangements, during the attachment/detachment cycle of the adhesion complex.
    • These rearrangements are hindered by antibodies against the C-terminal domain interfering with gliding,
      • Explains the specific neutralization mechanism deployed by antibodies against this domain
      • Suggests new ways to confront M. pneumoniae infections.

Cytokiny

  • IL-1beta, IL-2, IL-4, and IL-6 are elevated
    • In both the bronchoalveolar lavage fluid
    • In serum of these patients

Cytoplazma

  • Endoplazmatické retikulum
  • Ribozomy
  • Jaderná substance
    • Rozptýlená /v centru buňky [1]
  • Rigid cytoskeleton
  • Extracellular capsule
    • Facilitate adherence to the host cell [5]

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)


Glycerophosphodiesterase GlpQ

PMCID: PMC3178575; PMID: 21966272
  • Formation of hydrogen peroxide
    • A product of glycerol metabolism
  • Essential for host cell cytotoxicity
  • Phosphatidylcholine
    • Major carbon source available on lung epithelia
    • Utilization requires the cleavage of deacylated phospholipids
      • To glycerol-3-phosphate and choline
  • M. pneumoniae possesses two potential glycerophosphodiesterases
    • MPN420 (GlpQ)
    • MPN566
  • Only GlpQ is an active glycerophosphodiesterase.
  • Inactivation of the glpQ gene resulted in
    • Reduced growth in medium with glucose as the carbon source
    • Loss of hydrogen peroxide production when phosphatidylcholine was present
    • Complete loss of cytotoxicity towards HeLa cells
  • Reverted upon complementation of the mutant
  • GlpQ mutant strain exhibited a reduced gliding velocity
  • GlpQ acts as a trigger enzyme
    • Measures the availability of its product glycerol-3-phosphate
    • Uses this information to differentially control gene expression
  • M. pneumoniae is unable to cause any detectable damage to the host cells in the absence of GlpQ.
  • Major virulence determinant of M. pneumoniae is
    • hydrogen peroxide
      • Generated during the utilization of glycerol-3-phosphate,
        • Which might be derived from free glycerol or from the degradation of phospholipids.
  • Lecithin
    • Is the by far most abundant carbon source on lung epithelia
  • The pulmonary surfactant is composed of about 90% phospholipids and 10% proteins
    • Phospholipids play a major role in the nutrition of M. pneumoniae.
  • Glycerophospholipids
    • The major building blocks of the cell membrane in bacteria and eukaryotes
    • Degraded in several steps.
  • Fatty acids are cleaved from the phospholipids
    • Resulting in the formation of glycerophosphodiesters
    • Phosphate group of glycerol-3-phosphate is linked to another compound, called the head group
    • Eukaryotes
      • choline is by far the most abundant head group
      • Lecithin, the choline-containing phospholipid accounts for about 80% of all phospholipids in human lung cells
  • choline head group is cleaved
    • Due to the activity of a glycerophosphodiesterase
    • Resulting in the formation of glycerol-3-phosphate
      • Feed into glycolysis after oxidation to dihydroxyacetone phosphate
  • In M. pneumoniae
    • Catalyzed by the glycerol-3-phosphate oxidase GlpD
    • GlpD transfers the electrons to water
  • UgpC (MPN134), UgpA (MPN135), and UgpE (MPN136)
    • Putative ABC transport system for glycerol-3-phosphate
  • GlpF (MPN043)
    • Glycerol uptake facilitator
  • Glycerol kinase GlpK (MPN050) + glycerol-3-phosphate oxidase GlpD (MPN051)
    • Metabolize glycerol to the glycolytic intermediate dihydroxyacetone phosphate
  • Hydrogen peroxide formation by GlpD
    • Is crucial for the cytotoxic effects of M. pneumoniae
  • GlpQ (MPN420) and MPN566
    • Encode two paralogous glycerophosphodiesterases
    • May be able to metabolize glycerophosphocholine to glycerol-3-phosphate and choline
  • Enzyme generates choline, which in turn is used for the biosynthesis of the bacterial lipopolysaccharide layer - a major virulence determinant of Gram-negative bacteria

Glykany

  • Mycoplasmas can scavenge host structures, such as host glycans, for decoration of their own surface glycolipids to avoid detection

Metabolismus

  • Slower growth
  • Ferments glucose
  • Absorbs erythrocytes in the growing colonies
  • Reduces tetrazolium when grown aerobically or anaerobically [8]

Mycoplasmas secrete some of their own enzymes

Generation of hydrogen peroxide and superoxide radicals

Direct membrane-membrane interactions

Hide and survive

Suppress host cell responses

Host cell DNA fragmentation

  • M. penetrans can induce or enhance apoptosis of peripheral mononuclear cells
  • Sign of this is DNA fragmentation
    • DNA ladder formation seen after electrophoretic separation
    • Catalyzed by endogenous Ca2+, Mg2+-dependent endonucleases
  • M. penetrans endonuclease (p40)
    • Identified as a pathogenic mycoplasma determinant
  • www.scirp.org/journal/paperinformation.aspx?paperid=95720
  • Pathogenic mycoplasma-released nucleases
    • May also be involved in secondary necrosis
      • Apoptosis (chromatin condensation)
      • Necrosis (loss of membrane integrity with organelle swelling)
  • Cell death was accompanied by
    • Oligonucleosomal DNA fragmentation
    • Loss of chromosomal DNA
  • www.scirp.org/journal/paperinformation.aspx?paperid=95720

Purinergic receptor P2X7

  • A ligand-gated ion channel, plays a crucial role in inflammasome activation
    • During microorganism infection
    • By permitting the rapid efflux of potassium ions

Depletion of the mouse P2x7 gene

  • Will significantly damage the immune system
  • Increase infection.
  • Can be linked to respiratory infections such as MPP

Reprodukce

  • Exclusively parasitizes humans [5]
  • Does not need a host cell for replication [6]
  • Dependence upon the host for survival [5]
  • Reproduction depends upon attachment to a host cell
  • Binary fission
    • Temporally linked with duplication of its attachment organelle
      • Migrates to the opposite pole of the cell during replication and before nucleoid separation [5]
  • Mutations that affect the formation of the attachment organelle
    • X motility
    • X cell division
    • Reduce the ability of M. pneumoniae cells to adhere to the host cell [5]

Zánět plicní

IL-12 knock out

  • M. pneumoniae induces a Th1 cytokine response in the lungs
    • Associated AO, AHR, and lung inflammation
  • IL-4
    • Key role in Th2 signaling
  • IL-12
    • Pivotal role in the Th1-mediated immune response
    • IL-12 is a heterodimeric cytokine
    • Two subunits (p40 and p35)
    • Produced by antigen-presenting cells
      • Phagocytes
      • Dendritic cells
    • Targets natural killer and the T cells
      • Production of gamma interferon
  • Knockout IL-12 vedlo k dřívější úzdravě a menším škodám v plicích
  • europepmc.org/backend/ptpmcrender.fcgi?accid=PMC1828434&blobtype=pdf
  • NK cells in the IL-12 KO mice
    • Were only partially stimulated
    • IL-12 is a primary trigger for the NK cell activation
  • Production of pulmonary IFN-g significantly depressed by the absence of IL-12
    • Tnf alfa taky poklesl
      • Associated with M. pneumoniae disease severity
      • Decreased histologic lung inflammation in the IL-12 KO mice
    • Decrease in IL-10 in the IL-12 KO mice
      • Autoregulatory inhibitory mechanism of IL-10 was not engaged.
  • europepmc.org/backend/ptpmcrender.fcgi?accid=PMC1828434&blobtype=pdf

IFN- KO mice

  • Concentration of Mycoplasma in BAL samples was higher than in the infected WT

NK cells were depleted by anti-asialo GM1 antibody in IFN- KO mice



Thiopurines

  • Especially mercaptopurines
  • First line drugs for the treatment of acute leukemia since the 1950s
  • Treatment of inflammatory bowel disease

6-TG and 6-MP

  • Cytotoxicity through incorporation into DNA as deoxy-6-thioguanosine
  • Metabolized to deoxy-6-thioguanosine triphosphate
    • Via the purine salvage pathway initiated by HPRT
  • Thiopurine methyl transferase is a key enzyme in converting mercaptopurine to its cytotoxic metabolites
    • Can either inhibit purine nucleotide biosynthesis or incorporate into DNA or RNA
      • Causing DNA damage and cell death
  • Mpn does not possess the essential enzymes to convert mercaptopurine to the cytotoxic thioguanine nucleotides (methyl thiopurine nucleotides)
    • Inosine monophosphate dehydrogenase
    • Thiopurine methyl transferase
  • This may explain why 6-MP did not inhibit Mpn growth.

Other accessory proteins - inhibice

HMW1, HMW2, and HMW3 and A, B, and C

  • Collectively maintain the proper distribution and/or disposition of the adhesions in the mycoplasma membrane


Adheze na hostitelskou buňku

  • Respiratory epithelial cells
  • Erythrocyte
  • Urogenital lining cell [5]
  • Attachment organelle [5]
    • Polar, electron dense and elongated cell extension
      • Motility
      • Cytadherence
    • Electron-dense core with a trilaminar membrane [8]
  • Central filament
  • Surrounded by an intracytoplasmic space
  • Number of adhesins and structural and accessory proteins
    • At the tip of the organelle [5]
    • Accessory proteins - confer structure and adhesin support
      • HMW1–HMW5
        • Loss of HMW1–HMW3 = avirulence [5]
      • P30
        • Mutacemi / protilátkami - incapable of adhering to host cells [5]
        • Multiple lobes and a rounded shape
      • P56
      • P90 [5]
    • Involved directly in attachment
      • P1
        • trypsin-sensitive protein [5]
        • Highly clustered on the surface of the attachment organelle tip in virulent mycoplasmas [5]
        • Required for the attachment of M. pneumoniae to the host cell
        • Major component in cytadherence and motility
        • Mutations in P1 or degradation by trypsin treatment yield avirulent M. pneumoniae cells [5]
        • Important for pathogenesis [8]
        • Main antigens, with specific antibody production by the host [8]
      • P30
      • P116 [5]
  • Eukaryotní buňky - adeherence na povrch via
    • Sialoglycoconjugates
    • Sulfated glycolipids
    • Glycoproteins
    • Fibronectin
    • Neuraminic acid receptors
  • Lectins on the surface of the bacterial cells
    • Capable of binding oligosaccharide chains on glycolipids and glycoproteins to facilitate attachment [5]

Arginine deaminase

  • Growth-inhibitory enzyme derived from mycoplasmas
  • Inhibits the growth of human T-cells and T-lymphoblastoid cell lines
  • Arginine deaminase can suppresses
    • IL-2 production
    • Receptor expression in T-cells stimulated by non-specific mitogens
    • Inducing the morphologic features of dying cells
      • DNA fragmentation seen during apoptosis
  • Possible marker for M. pneumoniae infections
  • www.scirp.org/journal/paperinformation.aspx?paperid=95720

Buňky v BAL

  • High percentages of neutrophils and lymphocytes
    • Are present in the bronchoalveolar lavage fluid in patients with Mycoplasma pneumonia


Induce proinflammatory cytokine gene expressions

  • IL-1beta, IL-6, TNF-a, or IFN-g
  • Similar effects on human peripheral blood mononuclear cells
    • Jako EBV-positive lymphoblastoid cell lines
  • Respiratory epithelial cells
    • Main targets of M. pneumoniae
    • Secrete chemoattractant and proinflammatory cytokines
  • journals.asm.org/doi/10.1128/iai.70.7.3649-3655.2002

Cytotoxicity and organismal effects

  • Local disruption of tissue and cell structure along the respiratory tract epithelium
    • Close proximity to host cells
  • Loss of cilia
  • Reduction in metabolism, biosynthesis, and import of macromolecules
  • Infected cells may be shed from the epithelial lining
  • Community Acquired Respiratory Distress Syndrome (CARDS) toxin
    • Most likely aids in the colonization
    • Leading to inflammation
    • Airway dysfunction [5]
  • Formation of hydrogen peroxide
    • Key virulence factor in M. pneumoniae infections [5]
    • Loss of ciliar activity and finally to epithelium alterations [8]
  • Attachment of M. pneumoniae to erythrocytes
    • Permits diffusion of hydrogen peroxide from the bacteria to the host cell without detoxification by catalase or peroxidase
    • Injure the host cell by
      • reducing glutathione
      • Damaging lipid membranes
      • Causing protein denaturation [5]
  • Lactoferrin acquisition
    • hydroxyl radical
    • superoxide anion
    • peroxide formation
  • Common symptoms like
    • Coughing
    • lung irritation
    • may persist for months after infection has subsided [5]
  • Local inflammation and hyperresponsiveness
    • By infection induced cytokine production
    • Associated with chronic conditions such as:
      • Bronchial asthma (asthma related microbes) [5]
      • Progression of symptoms in cystic fibrosis and COPD [5]

17 enzymes in nucleotide biosynthesis

  • Identified in the Mpn genome

15 are essential

  • TK, HPRT, APRT and UPRT are essential for Mpn survival

Hypoxanthine guanine phosphoribosyltransferase (HPRT)

  • Nucleobases are salvaged through hypoxanthine guanine phosphoribosyltransferase (HPRT)

Adenine phosphoribosyltransferase (APRT)

  • Nucleobases are salvaged through hypoxanthine guanine phosphoribosyltransferase (HPRT)

Uracil phosphoribosyltransferase (UPRT)

  • Nucleobases are salvaged through hypoxanthine guanine phosphoribosyltransferase (HPRT)

Non-essential enzymes

Thymidylate synthase (TS)

  • Catalyses the reductive synthesis of thymidylate from uridylate
  • Is not since thyA mutant Mpn strain that lacks TS is viable

Evade the immune system

  • Membrane (in particular the high cholesterol contents)
    • High proportion of cholesterol, from 35 to 50% of the total lipid fraction
    • Constitutes about one third of the final membrane and about half of the lipid components of the membrane
    • Allows the mimicking of the host membrane
  • Lack of a cell wall
  • www.ncbi.nlm.nih.gov/pmc/articles/PMC7584665/

IL-17

  • Growing body of evidence demonstrates that IL-17 plays an important role in respiratory mycoplasma infection
  • Associates with the pathologic outcomes of infection
    • Pneumonitis and asthma
  • Particular Th17 cells in the lung
    • Secrete IL-17 during respiratory mycoplasma infection
  • onlinelibrary.wiley.com/doi/pdf/10.1111/imm.13346
  • IL-17 as the signature cytokine of a of CD4+ T helper 17 (Th17) cells
  • Activated by the IL-12 family cytokine IL-23
  • Transcription factor RAR-related orphan receptor gamma (RORgt)
    • Master regulator of Th17 cells
  • IL-17 in the lung plays essential roles in
    • Protecting against bacterial and fungal inf.
    • Maintaining the barrier function of epithelium and mucosa
    • Induction of granulocyte colony-stimulating factor (G-CSF), CXCL1 and CXCL8 (IL-8)
      • Neutrophil accumulation and activation
  • Together with IL-22, IL-17
    • Increases the expression of antimicrobial peptides
      • B-defensins, S100A8 and lipocalin 2
  • IL17 can also activate
    • Metalloproteinases
    • Inflammatory cascades closely associated with pneumonitis and asthma
  • Vital role of IL-17 in mediating both protective and pathogenic effects during mycoplasma infection

Acute M. pneumoniae infection murine model

  • M. pneumoniae infection dramatically enhanced the expression of IL-23
    • By alveolar macrophages (MΦ)
  • IL-17 and IL-17F in lung tissues
    • Increase in neutrophil recruitment
  • IL-23 neutralization
    • Caused a significant reduction in M. pneumoniae-induced IL-17 protein
    • Reduction in neutrophil recruitment,
    • reduction of M. pneumoniae clearance
  • IL-23 increases expression of IL-17
    • IL-17F in pulmonary CD4+ T cells
      • Important role of Th17 cells/IL-17 in M. pneumoniae infection

Chronic respiratory infection in mice with M. pulmonis

  • Th17 cells is significantly increased
  • IL-17-mediated neutrophils are essential for optimal clearance of M. pulmonis
  • Is impaired in C57BL/6 mice depleted of Gr-1+ cells or in
  • M. pneumoniae extract could induce a strong IL-17-associated inflammatory reaction
    • In mouse lung by recruiting neutrophils to the lungs
    • IL-17A exists in the gross lung lesions of Mycoplasma mycoides-infected cattle

Model of Mycoplasma bovis (M. bovis) infection

  • Induced Th17 responses are not protective
  • Virulent M. bovis-induced pathogenesis is associated with enhanced Th17 responses
  • Neutralizing IL-17A after infiltration of the lungs by T cells
    • Can reduce disease severity during M. pulmonis infection
    • But not in disease-resistant C57BL/6 mice
  • IL-17A neutralization did not further reduce disease
  • IL-17A worsens respiratory pathology
    • Mainly by recruiting neutrophils to the lung
    • Myeloperoxidase,
    • Matrix metalloproteinases and elastase
<4>High level of IL-17 in children with M. pneumoniae pneumonia

Cardiovascular extrapulmonary manifestations

  • Higher frequency of Th17 cells
  • Th17 cells/IL-17 may be involved in the clearance of M. pneumoniae
  • But excessive Th17 responses may contribute to the immunopathological damage
  • IL-23 may also drive IL17+ Th17 cells in patients with M. pneumoniae infection.
  • MiR-29c may inhibit Th17 responses in children with M. pneumoniae pneumonia
    • By targeting the costimulatory molecule B7-H3

Th17/Treg ratio

  • Involved in ninflammatory and autoimmune diseases
  • Higher in the patients with refractory M. pneumoniae
  • Closely related to the severity of illness
  • Tregs and IL-10
    • Suppression of IL-17A production

Depletion of CD25+ cells

  • Increases disease severity caused by M. pulmonis infection in BALB/c mice

CD4+CD25+ Tregs

  • Could promote IFNg+ and IL-17+ mycoplasma-specific CD4+ T-cell responses
  • Treg function was profoundly impaired during the acute stage of M. pneumoniae
  • In resolution stage
    • The Treg dysfunction was associated with an increase in IL-17A+ Tregs and Th17 cells
  • Th17 cell development
    • Dependent on the high level of IL-6 produced by the proinflammatory monocytes by TLR2 pathway
  • M. pneumoniae infection-induced Th17 cells/IL-17
  • Proportions of Th17 cells
    • Higher in bronchoalveolar lavage in M. pneumoniae-associated bronchiolitis obliterans
  • IL-17 may be critical for the clearance of mycoplasma infection in the early infection
  • Excessive Th17 responses contribute to the late mycoplasma pathology

Depletion of CD8+ T cells

  • Caused dramatically more severe pulmonary disease during M. pulmonis infection
  • Mycoplasma-specific CD8+ T-cell response is critical in regulating immunopathologic reactions
    • Higher percentage of Tc17 cells is found from patients with M. pneumoniae-associated bronchiolitis obliterans
      • Significantly correlated with reduced FEF25%–75% predicted values

Regulatory CD4+CD25+ T Cells

Th2 cell responses

  • Promote immunopathology
  • May also promote persistence of infection

Th1 cells

  • Confer resistance to infection

regulatory T cell population

  • Important role in controlling damaging immune responses in mycoplasma respiratory disease
  • Does not contribute to persistence of infection
  • Preferentially dampens Th2 cell-mediated inflammatory responses
    • Independent of IL-10 or TGF-b characteristic of “classic” Treg cells.
  • journals.plos.org/plosone/article?id=10.1371/journal.pone.0155648

Immune response

  • Intracellular localization
  • Change the composition of its cell membrane to mimic the host cell membrane
    • Avoid detection by immune system cells
    • Can also result in autoimmune responses in several organs and tissues [5]
  • Number of protein and glycolipid antigens that elicit immune responses
  • Eosinophil cationic protein (ECP)
    • Found to be increased [6]
    • May play a role in damage to the respiratory epithelium and accelerated hypersensitivity in the respiratory system [6]

Imunomodulace

  • Cytokine production
  • Direct effects on macrophages
  • B and T cells
  • Glial cells [8]
  • Activation of host-defense mechanisms [8]
  • Exacerbate disease through lesion development [8]
  • Related superantigen-like molecules
    • May exist in mycoplasmas of human origin
      • Triggering autoimmune
      • Other inflammatory pathologies [8]

Intracellular localization

  • Cell membrane components, like cholesterol, may facilitate fusion
    • Mimic the host cell membrane [5]
  • May produce chronic or latent infections as M. pneumoniae
    • Capable of persisting
    • Synthesizing DNA
    • Replicating within the host cell
    • Even after treatment with antibiotics [5]
  • Difficulty in completely eliminating M. pneumoniae infections in afflicted individuals [5]

Membrána

  • Někt. + další obal jako hutné pouzdro
  • Někt. povrchové ostré výběžky
    • Asi adhezivní
    • Těsná adheze nutná pro vpravení nukleáz a dalších enzymů do nitra napadené buňky
      • Získání produktů enzymatického štěpení
  • Nemají buněčnou stěnu jako ostatní bakterie
  • X syntézy peptidoglykanu
    • Naprosto rezistentní vůči antibiotikům blokujícím syntézu bakteriální stěny
      • Včetně penicilinu [1]
    • Importance in maintenance of osmotic stability to avoid desiccation [5]
    • susceptible to lysis by hypotonic solutions [8]
  • The only bacterial cells that possess cholesterol in their cell membrane
    • Obtained from the host [5]
  • M. pneumoniae more genes that encode for membrane lipoprotein variations than other mycoplasmas
    • Associated with its parasitic lifestyle [5]

Mycoplasma TK

  • Essential enzyme while TS is not
  • Expression of TK in Mpn was correlated with Mpn growth and DNA synthesis
  • Upregulation of TK activity was observed in an Mpn strain lacking TS activity
  • Phosphorylated products of TFT and 5FdU by TK
    • Irreversibly inhibit TS activity via covalent binding to the enzyme
    • Down regulation of TS activity leads to upregulation of TK activity
      • Similar to what was observed with the thyA mutant
  • Increased salvage of dT due to the induction of TK activity
    • Leads to higher level of dTTP, an allosteric regulator of purine nucleotide reduction by ribonucleotide reductase.
  • Inhibition of ribonucleotide reductase activity by high level of dTTP
    • Led to decreased uptake and incorporation of labelled nucleobases
    • May result in imbalance in nucleotide pools
  • High TK activity facilitates the phosphorylation of TFT and 5FdU
    • And accumulation of TFT-TP and 5FdUTP that may affect the integrity of DNA and lead eventually to cell death
  • Both TFT and 5FdU inhibited the growth of both wild type and the thyA mutant strain to the same extent
  • TK activity is upregulated by TFT and 5FdU,
    • TK plays an important role in growth inhibition observed with these compounds.

Cytokiny

  • Pathogenic mycoplasmas can also induce B-cell differentiation
  • Trigger the secretion of pro-inflammatory cytokines
    • Interleukin-1beta (IL-1beta), IL-2, IL-6, IL-8, tumor necrosis factor-alpha
    • Granulocyte macrophage-colony stimulating factor (GM-CSF) from cells
  • Release of inflammatory cytokines in vivo
    • Is predictive of refractory mycoplasmal infections in children

Mycoplasma-derived lipopeptides

  • Can directly stimulate host response cells, such as macrophages

Nitric oxide release

  • By macrophages
    • Indicator of immune stimulation

Secret soluble factors

  • Can activate and stimulate proliferation or inhibit the growth and differentiation of immune competent cells

Proliferativw responses

  • M. penetrans can induce significant proliferative responses in peripheral blood mononuclear cells
    • Associated with the expression of surface markers of lymphocyte activation
  • Observed in lymphocytes (both CD4+ and CD8+ T lymphocytes) from healthy donors
    • As well as from HIV-infected subjects at different stages of disease progression
  • Pathogenic mycoplasmas have evolved with the ability to modulate and interfere with host responses.

Mycoplasma immune modulation

  • Secretion of immune-modulating substances stimulated by pathogenic Mycoplasma species

M. fermentans-derived lipopeptide

  • Potent activator of macrophage function
  • M. fermentans-derived lipoproteins
    • Can interfere with the interferon gamma-dependent (IFN-?-dependent) expression of MHC class II molecules on macrophages
  • Mycoplasma fermentans a released lipoprotein
    • Can stimulate the induction of monocyte cytokines and chemokines
  • Spiralin - mycoplasma lipoprotein
    • Can stimulate the in vitro proliferation of human peripheral blood mononuclear cells and murine splenocytes
      • Secretion of proinflammatory cytokines
        • TNFalpha, IL-1 and IL-6
    • Can also induce the maturation of murine B-cells
  • Similar to other immune-modulating lipoproteins secreted by other pathogenic bacteria
    • Contributes significantly to patient morbidity.
  • Mycoplasmas can evade immune recognition and destruction
    • By undergoing rapid surface antigenic variations
  • Chronic nature of mycoplasmal infections
  • Slow-growing microorganisms like mycoplasmas

Necroptosis

  • Bacterial cellular components, metabolites and toxins released from Mycoplasma pneumoniae
    • Can induce immune-pathological damage in the host tissues
  • Ca2+-dependent cytotoxic nuclease (encoded by MPN133) produced by Mycoplasma pneumoniae
    • Can lead to apoptotic-like programmed cell death in the host
  • Mycoplasma pneumoniae lipoproteins
    • Can be recognized by TLRs,
      • Which stimulate the release of pro-inflammatory cytokines including TNF-a, IL-1b, and IL-6
  • Mycoplasma hyopneumoniae
    • Induces high levels of TNF-a production in alveolar macrophages
  • Excessive TNF-a secretion activating the necroptotic signaling pathway
    • Through RIPK1, RIPK3, and MLKL-dependent mechanisms, causing lung inflammation
  • www.mdpi.com/1999-4915/16/1/94

  • Absence of vitamins and lipids synthesis
  • Inability to detoxify metabolic waste (e.g., hydrogenperoxide)
  • Primarily ferments glucose
    • And uses most of its energy (71 to 88%) for non-growth associated tasks
      • Většinou na vypumpování H+ z buňky ven
      • Mohlo by to znamanat, že nízké pH v prostředí jim udělá více obtíží ?
      • O kyslém pH je známo, že potlačuje růst mnohých mikroorganismů...
  • Monocarboxylic acids transporters discovery
    • no lactate or acetate proton symporter were identified to be present in M. pneumoniae genome
  • Low oxygen
    • Pushes the lactate dehydrogenase towards the production of lactate, b
    • Critically affects the whole glycolytic pathway
      • Reduction of sugar uptake and ATP production
      • More NADH is consumed and less is regenerated by the GADPH enzyme
    • Production and accumulation of
      • Lactate and acetate augments
      • Enhancing toxicity and acidifying the medium when exported
    • Doubling time of M. pneumoniae in batch culture increases over time
      • Due to the decrease in media pH11
      • Accumulation of toxic waste compounds - acetic and lactic acid.
  • Cells growing in an acidified medium
    • Different membrane composition
      • Unclear whether the adaptability of M. pneumoniae membrane in genome-coding function or is it just a chemical-physical response of the lipid bilayer to pH changes or if it is a combination of these two aspects
  • De-acidifying the cytosol
    • A cytosolic pH decrease is equivalent to an increase in the H3O+ intracellular concentration
      • Might alter reactions involving H+ as reactant
    • High energy maintenance costs and reduced growth rate
  • Production of M. pneumoniae in serum-free media is a major challenge
    • Requirement for sterol components in serum
    • Required daily changes of the medium
  • M. pneumoniae preferentially incorporates in the membrane sphingomyelin with less than 20 carbons-fatty acid chains. M. pneumoniae is a fatty acid and sterol auxotroph, but it can assemble phosphatidic acid, diacylglycerol, phosphatidylglycerol and glycolipids from provided fatty acids in the medium
  • Glycolipids constitute a range of 5–15% of the total membrane lipids41, while the remaining percentage should be constituted by phosphatidylglycerol; phosphatidic acid and diacylglycerol,
  • M. pneumoniae tends to selectively incorporate palmitic acid over stearic acid in the membrane30, with a preference for saturated fatty acids, suggesting oleic and linoleic acids are incorporated in a lower proportion with respect to the amount found in the media.
  • Sphingomyelin, on the other hand, is in our model directly incorporated into the membrane without modifications
  • www.ncbi.nlm.nih.gov/pmc/articles/PMC7584665/

VĂ­ce druhĹŻ

  • M. pneumoniae is generally considered to be a rather homogeneous organism
  • Naturally occurring variations in the primary structure of the P1 adhesin
    • M. pneumoniae epidemiology,
  • Variant sequences may be degenerate repeats of regions of the P1 coding gene throughout the M. pneumoniae genome
  • Variation in P1 might be associated with short-term immunity to individual subtypes
    • Development of subtype-specific antibodies
    • Frequent re-infections with another subtype

Generation of peroxide and superoxide radicals

  • Endogenous toxic oxygen molecules generated by the host to induce oxidative stress
  • Superoxide anions produced by M. pneumoniae act to inhibit catalase in host cells
    • Reducing the enzymatic breakdown of peroxides produced endogenously and by the mycoplasma
      • Rendering the host cell more susceptible to oxidative damage.

HPr kinase/phosphorylase (HPrK)

  • One of the nine known regulatory proteins in the M. pneumoniae genome
    • Key regulator of carbon metabolism in many Gram-positive bacteria
  • HPrK phosphorylates/dephosphorylates the HPr protein
    • Bacterial phosphotransferase system on a serine residue
    • In response to the nutrient status of the cell
  • HPrK activity at low ATP concentrations
    • Activation of this enzyme requires glycerol derived from the phospholipids of the host-cell membrane
    • May be linked to peroxide production and virulence.
  • hydrogen peroxide activates cells
    • Through selective inactivation of protein tyrosine phosphatases

Community-acquired respiratory distress syndrome toxin (CARDS TX)


Homologs of proteins associated with the pertussis toxin S1 subunit

  • Ability to be translocated from the pathogen to the host-cell cytoplasm
    • C-terminal moiety of CARDS TX is novel and the protein is immunodominant
      • Ability to be translocated

Adhesins to extracellular host proteins, such as

  • Fibronectin
  • Mucin
  • Cells of the respiratory epithelium typically
    • Lose their cilia,
    • Appear vacuolated,
    • Show a reduction in oxygen consumption, glucose utilization, amino acid uptake and macromolecular synthesis,
    • Resulting in exfoliation
  • Damage to cells of the upper respiratory tract
    • Ensuing local inflammatory response is reflected
      • Pharyngitis, hoarseness, coryza, earache, conjunctivitis and cervical adenopathy.

Mycoplasmas reaching the lower respiratory tract

  • Are opsonized by antibody and complement
  • Phagocytized by activated macrophages
  • Results in an inflammatory exudate that also includes neutrophils and lymphocytes
  • Complement-mediated cytolysis
    • May then play a role in limiting the growth of the mycoplasma.

Immune response

  • May not eliminate the organisms, allowing long-term carriage in some persons
  • Congenital hypogammaglobulinemia may experience chronic mycoplasmal respiratory infections
    • Sometimes accompanied by dissemination of infection to extrapulmonary sites

Adheze a dál

  • M. pneumoniae stimulates production of IL-4 by mast cells in co-culture experiments
    • Dependent upon the presence of
      • Sialic acid residues on the target cell membrane
      • P1 adhesin components of the attachment organelle
      • Expression of the heavily sialylated FceRI a-chain by the mast cell
  • Mycoplasma adherence to surface sialoglycoproteins results in
    • Cellular activation through the normal receptor signaling mechanisms
  • Hydrogen peroxide produced by M. pneumoniae
  • By reversibly oxidizing an essential cysteine in the catalytic site of mast cells inhibit
    • protein tyrosine phosphatases
    • Lipid phosphatase
    • Tensin homolog
  • M. pneumoniae also directly activates and induces cytokine production from
    • Peripheral blood leukocytes,
    • Respiratory epithelial cells
    • Macrophages
  • TNF-a, IL-8 and IL-1b mRNA are
    • By cultured human lung alveolar type II pneumocytes infected with M. pneumoniae
    • Adherence to human airway epithelial cells
      • Production of cytokines
      • Recruitment of lymphocytes
      • Other inflammatory cells
  • Activation of phagocytes for TNF-a production
    • Does not appear to require cell contact
    • Can be induced with mycoplasma culture supernatants
  • Activation of Toll-like receptor (TLR)-1 and -2
    • By mycoplasma-derived lipopeptides
  • Mycoplasma cell membrane-derived dipalmitoylated lipoprotein subunit b
    • Of F0F1-type ATPase
    • Activate nuclear factor-kB through TLR-1, -2 and -6
      • In a human monocytic cell line, THP-1
  • F0F1-type ATPase in a rodent model
    • Important in the induction of the inflammatory response
    • Through induction of chemokines and inflammatory cytokines followed by neutrophil infiltration

Cytokines + lymphocyte activation

  • May either minimize disease
    • Host defense mechanisms +
    • Subsequent elimination
  • May exacerbate disease
    • Immunologic hypersensitivity
    • Worsening damage to the respiratory epithelium
    • Inflammatory response +++
  • The more vigorous the cell-mediated immune response and cytokine stimulation
    • The more severe the clinical illness and pulmonary injury becomes
  • Persistence of M. pneumoniae in the host
    • Leads to the provocation of ineffective immune-mediated inflammatory responses
      • Regulated by opposing T-cell activities

Autoimmunity & extrapulmonary manifestations

  • Skin and the nervous, cardiovascular, renal, gastrointestinal, musculoskeletal and hematologic systems
    • In as many as 25% of infected persons

Neurologic complications

  • The most common extrapulmonary manifestations of M. pneumoniae infection
  • Encephalitis
    • M. pneumoniae was the most common agent identified
      • Mainly by serological means
      • PCR assays on cerebrospinal fluid were rarely positive
  • Clusters of encephalitis
    • May also occur during epidemics of M. pneumoniae respiratory illness
      • Sometimes overshadowing the respiratory problems
  • Cross-reactive antibodies to the brain and other neurologic structures
    • Manifestations following M. pneumoniae infection
    • Molecular mimicry with carbohydrate moieties of the
      • Abundant glycolipids in the M. pneumoniae membrane
      • Lipoglycan capsule

Anti-GM1 and galactocerebroside antibodies

  • Primary autoantibodies implicated in the ascending paralysis of Guillain-BarrĂ© syndrome
  • In encephalitis associated with M. pneumoniae
  • Cytokines IL-6 IL-8 can be elevated in the cerebrospinal fluid in encephalitis
    • Appear to be important mediators of inflammation

Invasion of the CNS itself

  • RNA can detected in brain tissue
    • By nucleic acid hybridization
    • Demonstrated in cerebrospinal fluid by PCR and culture
  • Case of fatal encephalitis
    • Antigens were immunohistochemically detected in histopathologically involved areas of a brain biopsy and at autopsy

Autoimmune hematologic phenomena

Cold agglutinins

  • Frequently occur following M. pneumoniae
    • Formerly a primary diagnostic tool
    • Occasionally induce a transient brisk hemolytic anemia
    • Most often in children
    • Termed paroxysmal cold hemoglobinuria
  • Primarily of the IgM isotype
  • Antigen is primarily of the Ii blood group
    • Branched (I) or linear (i) polymer of N-acetylgalactosamine
    • Found on surface glycoproteins or glycolipids of erythrocytes and other cells
  • Antigen is not found in the M. pneumoniae membrane
    • It acts as a surface receptor for the organism
    • Thereby apparently rendered immunogenic.

Presence of M. pneumoniae

  • In blood,
  • Synovial fluid,
  • Cerebrospinal fluid,
  • Pericardial fluid
  • Skin lesions
  • Documented by PCR and/or culture

Adult rheumatoid arthritis

Juvenile idiopathic arthritis

Crohn's disease

Athma & chronic lung disease

  • Ability to induce chronic disease states
    • Clearance of the organism is extremely difficult
  • Intracellular localization,
  • Immunomodulatory effects
  • Surface-antigen variations
  • Pathogenesis of asthma was suggested more than 30 years ago
  • Support for this theory is now strong
  • In pathogenesis as well as in exacerbation of acute attacks
  • Can be isolated in higher prevalence from asthmatics than from healthy persons;
  • Macrolide antibiotics can result in improvement in pulmonary function in asthma
  • Follow-up studies in children
    • Prolonged airway dysfunction consistent with a persistent infection;
  • M. pneumoniae induces a number of inflammatory mediators implicated in the pathogenesis of asthma
    • IgE,
    • Substance P
    • Neurokinin 1,
    • IL-5
  • Models of chronic respiratory infection in mice
    • Organisms can produce pneumonia
    • Stimulate cytokine production,
    • Airway hyper-responsiveness resembling chronic asthma
    • Th2-dominant airway inflammatory process that potentiates organism survival in the lungs
  • Facilitate alterations in local respiratory immunity
    • Activation or suppression of pulmonary macrophages or T-cells
  • Some mycoplasmas have the capacity to alter the
    • Structures and functions of host pulmonary cells
      • Activation and inflammation of lung epithelium and endothelium
  • www.scirp.org/journal/paperinformation.aspx?paperid=95720
  • Adhesion to respiratory epithelial cells
  • Unique gliding motility
  • Induction of pathological hyper-stimulation of local host cellular response mechanisms appear to be important
  • Mimic some of the structures of pulmonary host cells
    • Antigenic similarities between important functional adhesion molecules of M. pneumoniae and various host cell surface molecules
      • Could be one of the factors responsible for hindering host recognition
      • Failure to protect against repeated mycoplasma pulmonary colonizations

  • The invasion of MP activates the body’s innate immune system
  • TLR2 on immune cells
    • Specifically bind to Mycoplasma pneumoniae
      • Inflammatory infiltration occurs
        • Pro-inflammatory cytokines (Cronin et al., 2012)
  • Recognition of pathogen-associated molecular patterns (PAMP)
    • Receptors on cells of the innate immune system
      • TLR1-10 (Takeuchi and Akira, 2010)
  • TLR2 and TLR4 specifically recognize MP
    • Through MyD88-dependent pathways
      • Recruitment, activation, and degradation of IRAK-1,
      • Degradation of the downstream protein I?B kinase (IKK)
      • NF-kB activation
      • Expression of IL-1b, IL-6, IL-8, and TNF-a (Lye et al., 2004; Shimizu, 2016; Chen J. et al., 2020).

Community-acquired respiratory distress syndrome toxin (CARDS)

  • Mycoplasma pneumoniae-released factor
  • ADP-ribosylating and vacuolating cytotoxin
  • Activates the NLRP3 inflammasome complex
  • Causes subsequent release of IL-1 and hyper-inflammation
  • Cause tissue damage and other pathologies
  • Mycoplasma toxin appears to cause
    • Pulmonary inflammation,
    • Cytokine release,
    • Significant airway dysfunction
    • May be responsible, in part, for respiratory failure
      • Fatal outcomes found in acute M. pneumoniae infections
  • www.scirp.org/journal/paperinformation.aspx?paperid=95720

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Poslední aktualizace: 28. 4. 2024 11:47:32