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

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

Mycoplasma pneumoniae Cytoskeletal Protein HMW2 and the Architecture of the Terminal Organelle

The terminal organelle of Mycoplasma pneumoniae mediates cytadherence and gliding motility and functions in cell division. The defining feature of this complex membrane-bound cell extension is an electron-dense core of two segmented rods oriented longitudinally and enlarging to form a bulb at the distal end. While the components of the core have not been comprehensively identified, previous evidence suggested that the cytoskeletal protein HMW2 forms parallel bundles oriented lengthwise to yield the major rod of the core. In the present study, we tested predictions emerging from that model by ultrastructural and immunoelectron microscopy analyses of cores from wild-type M. pneumoniae and mutants producing HMW2 derivatives. Antibodies specific for the N or C terminus of HMW2 labeled primarily peripheral to the core along its entire length. Furthermore, truncation of HMW2 did not correlate specifically with core length. However, mutant analysis correlated specific HMW2 domains with core assembly, and examination of core-enriched preparations confirmed that HMW2 was a major component of these fractions. Taken together, these findings yielded a revised model for HMW2 in terminal organelle architecture.Mycoplasma pneumoniae is a cell wall-less pathogen of the human respiratory tract causing community-acquired tracheobronchitis and atypical, or “walking,” pneumonia (38). Colonization of the respiratory mucosa is mediated in large part by the terminal organelle, a polar, tapered extension of the mycoplasma cell having a high density of receptor-binding proteins (4, 22, 28). The terminal organelle also constitutes the motor in gliding motility (5, 11), and its duplication precedes cell division (5, 12, 32).Ultrastructurally, the terminal organelle is defined by a characteristic electron-dense core consisting of a thick rod and a thin rod oriented longitudinally in parallel and capped by a terminal button at the distal end (4, 16, 17, 39, 41). The core and terminal button are elements of the mycoplasma cytoskeleton (triton shell), a complex network of proteins resistant to extraction with Triton X-100 (TX) (1, 7, 24), much like the cytoskeletal fraction of eukaryotic cells (18, 33, 34). The composition of the M. pneumoniae triton shell has been examined by using antibody probes (22) and by mass spectrometry (29), but the identities of proteins specific to the electron-dense core are largely unknown, although cores fail to assemble in the absence of cytoskeletal proteins HMW1 and HMW2, both of which localize to the terminal organelle (3, 31, 36).HMW2 is a large protein (1,818 residues) predicted to have a globular N terminus followed by 10 dimeric or trimeric coiled-coil domains interspersed with leucine zipper motifs (23) (Fig. ​(Fig.1).1). Spontaneously arising mutant I-2 lacks HMW2 due to a frameshift in the corresponding MPN310 open reading frame, which also encodes protein P28 at its 3′ end, in the same reading frame encoding HMW2 (6). Mutants C1 and H9 are similar to mutant I-2 but result from Tn4001 disruption of MPN310 (15, 23) (Fig. ​(Fig.1).1). The loss of HMW2 and the inability of these mutants to assemble a core are accompanied by an abnormal morphology, reduced levels of terminal organelle proteins HMW1, HMW3, P24, P28, P41, and P65, failure to localize the major adhesin P1 to the terminal organelle, and the loss of cytadherence (6, 20, 22, 31). Imprecise transposon excision from mutant C1 yielded excision revertant C1R1, having an in-frame deletion in MPN310 that truncates HMW2 and eliminates P28 (6) (Fig. ​(Fig.1).1). Analysis using immunofluorescence (40) or fluorescent protein fusions (3, 19) localizes HMW2 generally to the terminal organelle. Based on its localization, its requirement for core formation, and its deduced length relative to that of the core, we proposed previously that HMW2 is a major component of the electron-dense core and, with P28, may form bundles oriented longitudinally to yield the large rod of the core (3).Open in a separate windowFIG. 1.Structural features of the indicated wild-type (WT), engineered, and mutant HMW2 proteins. White boxes, predicted dimeric coiled coils; black boxes, predicted trimeric coiled coils; dark gray boxes, leucine zipper motifs; arrows, predicted N terminus of P28; black triangles, cysteine residues. The numbers above each diagram correspond to the coiled-coil regions, while the letters below correspond to the leucine zipper motifs.In the present study, we explored further the role of HMW2 in core formation relative to the current model, by which (i) HMW2 is predicted to orient with its N- and C-terminal domains at the ends of the large rod of the core and (ii) mutants producing shorter HMW2 proteins are expected to have correspondingly shorter cores. We report here the successful localization of HMW2 by immunoelectron microscopy (immuno-EM) and the ultrastructural analysis of electron-dense cores in several HMW2 truncation mutants, allowing us to correlate specific regions of HMW2 with normal core formation. Finally, we evaluated core enrichment following detergent and salt extractions, demonstrating that HMW2 was a major component of a core-enriched fraction (CEF). Alternative models for HMW2 in core architecture are considered, based on our observations.     

A 41-kDa variable surface protein of Mycoplasma gallisepticum has a counterpart in Mycoplasma imitans and Mycoplasma iowae

Abstract Mycoplasma gallisepticun, M. imitans and M. iowae are three morphologically similar avian Mycoplasma species, and M. gallisepticum and M. imitans have been shown to be antigenically related. Using a monoclonal antibody that binds to the previously described size- and phase-variant integral membrane surface protein PvpA of M. gallisepticum , we have identified in all three avian Mycoplasma species a 41-kDa surface antigen, which in M. gallisepticum and M. imitans was identified as peripheral membrane protein undergoing variation in expression among clonal isolates. Southern blot analysis using the pvpA gene as a probe demonstrated sequence homology with M. imitans and M. iowae genomic DNA and suggested that a pvpA -related gene that may encode the 41-kDa product exists in these two Mycoplasma species. These studies establish (i) that M. iowae is antigenically related to M. gallisepticum and M. imitans , (ii) that the three species share non-ribosomal gene sequences, and (iii) that peripheral membrane proteins contribute to Mycoplasma surface variation.     

P110 and P140 Cytadherence-Related Proteins Are Negative Effectors of Terminal Organelle Duplication in Mycoplasma genitalium

Background

The terminal organelle is a complex structure involved in many aspects of the biology of mycoplasmas such as cell adherence, motility or cell division. Mycoplasma genitalium cells display a single terminal organelle and duplicate this structure prior to cytokinesis in a coordinated manner with the cell division process. Despite the significance of the terminal organelle in mycoplasma virulence, little is known about the mechanisms governing its duplication.

Methodology/Principal Findings

In this study we describe the isolation of a mutant, named T192, with a transposon insertion close to the 3′ end of the mg192 gene encoding for P110 adhesin. This mutant shows a truncated P110, low levels of P140 and P110 adhesins, a large number of non-motile cells and a high frequency of new terminal organelle formation. Further analyses revealed that the high rates of new terminal organelle formation in T192 cells are a direct consequence of the reduced levels of P110 and P140 rather than to the expression of a truncated P110. Consistently, the phenotype of the T192 mutant was successfully complemented by the reintroduction of the mg192 WT allele which restored the levels of P110 and P140 to those of the WT strain. Quantification of DAPI-stained DNA also showed that the increase in the number of terminal organelles in T192 cells is not accompanied by a higher DNA content, indicating that terminal organelle duplication does not trigger DNA replication in mycoplasmas.

Conclusions/Significance

Our results demonstrate the existence of a mechanism regulating terminal organelle duplication in M. genitalium and strongly suggest the implication of P110 and P140 adhesins in this mechanism.     

Characterization of a major Mycoplasma penetrans lipoprotein and of its gene

Abstract A novel mycoplasmal species designated as Mycoplasma penetrans has been isolated recently from patients infected with human immunodeficiency virus. p35, a major antigen extracted from the membrane of this mycoplasma using Triton X-114 has been found to be a lipoprotein. After proteolytic treatment of p35, the sequence of one of the resulting peptides was determined and a corresponding oligonucleotide was deduced. Using this oligonucleotide as a probe the p35 gene was cloned and sequenced. Sequence analysis revealed an amino-terminal signal peptide with a potential acylation site which would result in a 35.3 kDa mature product. In addition, the p35 gene was followed by an open reading frame with a corresponding polypeptide partially homologous to p35, in particular to the N-terminus region.     

Genome sequence of Mycoplasma iowae strain 695, an unusual pathogen causing deaths in turkeys

Mycoplasma iowae is associated mainly with reduced hatchability in turkeys and is well known for the unusual ability of phenotypic variation in the Mycoplasma surface components as well as a relative resistance to heat, bile salts, and many antimicrobials. A subset of unique genes and a gene cluster responsible for these characteristics could be identified from the genome. Here, we report the first genome sequence of this species.     

IgA肾病患者血液、尿液及咽拭子标本中穿通支原体的分离检出

目的探讨IgA肾病患者血液、尿液及咽拭子标本中穿通支原体（Mycoplasma penetrans,Mp）的分离检出率以及与病理型别相关性。方法采用分离培养法,共计从26例IgA肾病患者血液、尿液及咽拭子标本及38例正常对照相应标本中进行穿通支原体分离检测,对培养阳性标本用穿通支原体套式PCR进行证实。结果在11例（42．3％）患者血液与尿液或（和）咽拭子中同时分离到穿通支原体,单独尿液或咽拭子标本阳性分别为1例（3．8％）与7例（26．9％）。26例IgA肾病患者血液、尿液及咽拭子穿通支原体的分离检出率分别为42．3％、23．1％与57．7％;与38例正常对照组血液、尿液及咽拭子检出0例、2例（5．3％）与7例（18．4％）相比较,差异有非常显著性（P〈0．01）,在正常对照组中无2种以上标本同时检出穿通支原体。结论穿通支原体在ISA肾病患者的血液、尿液与咽拭子标本中均有较高的检出率且与病理型别有一定的相关性。     

Morphology and Ultrastructure of Mycoplasma pneumoniae Spherules

During growth in fluid medium, most strains of Mycoplasma pneumoniae produce free-floating granules which become larger with time. We have called these granules "spherules." This study describes the morphological and ultrastructural features of M. pneumoniae strain AP-164 spherules, both free and in association with HeLa cells in cell culture. In thin section, spherules were composed of lobulated cells, connected together by membranes, and ring-shaped cells. The two-dimensional morphology observed varied according to the plane of section and to the age of the culture. In HeLa cell cultures, mycoplasmata were found attached to plasma membranes of cells; in sections, individual mycoplasmata were often aligned in radial apposition to the membranes. Mycoplasmata were not found intracellularly. The three-dimensional morphology of spherules was examined by the critical point method and by scanning electron microscopy. Both methods revealed lobulated forms, ring-shaped forms, and star-shaped forms. Treatment of the spherules with crude porcine pancreatic lipase effectively released large numbers of free organisms. Phosphotungstic acid preparations of these uncentrifuged forms revealed a morphology in agreement with the other methods used. Lobulated ring forms with "beaded" filaments were prominent. In respect to morphology, M. pneumoniae under our conditions resembles that of the type species M. mycoides.     

The complete genomic sequence of Mycoplasma penetrans,an intracellular bacterial pathogen in humans

The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans.     

Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease.

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and linear double-stranded DNA and a nuclease activity on RNA and single-stranded DNA. No exonuclease activity was found for this enzyme. This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.     

Disruption of a Dynamin Homologue Affects Endocytosis, Organelle Morphology, and Cytokinesis in Dictyostelium discoideum

The identification and functional characterization of Dictyostelium discoideum dynamin A, a protein composed of 853 amino acids that shares up to 44% sequence identity with other dynamin-related proteins, is described. Dynamin A is present during all stages of D. discoideum development and is found predominantly in the cytosolic fraction and in association with endosomal and postlysosomal vacuoles. Overexpression of the protein has no adverse effect on the cells, whereas depletion of dynamin A by gene-targeting techniques leads to multiple and complex phenotypic changes. Cells lacking a functional copy of dymA show alterations of mitochondrial, nuclear, and endosomal morphology and a defect in fluid-phase uptake. They also become multinucleated due to a failure to complete normal cytokinesis. These pleiotropic effects of dynamin A depletion can be rescued by complementation with the cloned gene. Morphological studies using cells producing green fluorescent protein-dynamin A revealed that dynamin A associates with punctate cytoplasmic vesicles. Double labeling with vacuolin, a marker of a postlysosomal compartment in D. discoideum, showed an almost complete colocalization of vacuolin and dynamin A. Our results suggest that that dynamin A is likely to function in membrane trafficking processes along the endo-lysosomal pathway of D. discoideum but not at the plasma membrane.     

HMW1 Is Required for Cytadhesin P1 Trafficking to the Attachment Organelle in Mycoplasma pneumoniae

Mycoplasma pneumoniae proteins HMW1-HMW3 collectively are essential for cytadherence, but the function or requirement for each has not been defined. Cytadherence mutant M6 lacks HMW1 because of a frameshift in hmw1 and produces a truncated adherence-associated protein P30 because of a deletion at the 3′ end of p30. Genetic manipulation of this mutant was used to evaluate the role of HMW1 in cytadherence. Mutant M6 was transformed with a recombinant transposon containing a wild-type p30 allele. Transformants synthesized both truncated and full-length P30, from the resident and recombinant alleles, respectively. However, these transformants remained hemadsorption negative, suggesting that HMW1 is required for cytadherence. Wild-type M. pneumoniae cells are generally elongated, tapering to form the attachment organelle at one end of the cell. The cytadhesin protein P1 is normally densely clustered on the mycoplasma surface at this differentiated terminal structure. However, both mutant M6 and M6 transformed with recombinant p30 had a striking ovoid morphology with no tapering at the tip structure, making the attachment organelle indistinguishable. Furthermore, protein P1 was randomly distributed on the mycoplasma surface rather than clustered at a polar location. In contrast, mutant M6 transformed with a recombinant transposon expressing the wild-type hmw1 allele exhibited a near-normal morphology and localized P1 to the attachment organelle. Significantly, M6 transformed with an hmw1 gene truncated slightly at the 3′ end failed to restore proper morphology or P1 localization to the attachment organelle, suggesting a functional importance to the C-terminal domain of HMW1.     

KCNQ Channels Show Conserved Ethanol Block and Function in Ethanol Behaviour

In humans, KCNQ2/3 channels form an M-current that regulates neuronal excitability, with mutations in these channels causing benign neonatal familial convulsions. The M-current is important in mechanisms of neural plasticity underlying associative memory and in the response to ethanol, with KCNQ controlling the release of dopamine after ethanol exposure. We show that dKCNQ is broadly expressed in the nervous system, with targeted reduction in neuronal KCNQ increasing neural excitability and KCNQ overexpression decreasing excitability and calcium signalling, consistent with KCNQ regulating the resting membrane potential and neural release as in mammalian neurons. We show that the single KCNQ channel in Drosophila (dKCNQ) has similar electrophysiological properties to neuronal KCNQ2/3, including conserved acute sensitivity to ethanol block, with the fly channel (IC₅₀ = 19.8 mM) being more sensitive than its mammalian ortholog (IC₅₀ = 42.1 mM). This suggests that the role of KCNQ in alcohol behaviour can be determined for the first time by using Drosophila. We present evidence that loss of KCNQ function in Drosophila increased sensitivity and tolerance to the sedative effects of ethanol. Acute activation of dopaminergic neurons by heat-activated TRP channel or KCNQ-RNAi expression produced ethanol hypersensitivity, suggesting that both act via a common mechanism involving membrane depolarisation and increased dopamine signalling leading to ethanol sedation.     

新伪尾柱虫腹皮层纤毛器微管胞器的形态和形态发生

应用荧光紫杉醇直接荧光标记法显示,腹毛目纤毛虫新伪尾柱虫(Pseudourostyla nova)腹皮层纤毛器微管胞器由口围带、波动膜、额腹横棘毛和左右缘棘毛等纤毛器微管及纤毛器基部附属微管组成.口围带基部含小膜托架及与托架相联系的肋壁微管,其中领部小膜托架间由"Λ"形微管相联接;额腹横棘毛基部含前纵微管束、后纵微管束、横微管束和周围微管束,其微管在不同棘毛基部的发达程度不一;缘棘毛基部含前纵微管束、后纵微管束.同时,对新伪尾柱虫纤毛器微管胞器的形态发生和生理改组过程进行了详细的追踪研究,并对细胞皮层的额腹棘毛定位及组成特征进行了补充报道.此外,发现形态发生末期新纤毛器微管形成时,残存部分老额棘毛、横棘毛和缘棘毛,此后老结构逐渐被吸收.结果表明,新伪尾柱虫的纤毛器基部微管具有其种的特异性,新纤毛器微管分化过程中老结构可能具有定位和物质贡献作用.