Mollicutes: Molecular Biology and Pathogenesis | Book
Caister Academic Press
Glenn F. Browning and Christine Citti Asia-Pacific Centre for Animal Health, Faculty of Veterinary Science, The University of Melbourne, Parkville Victoria 3010, Australia and INRA, École Nationale Vétérinaire de Toulouse, 31076 Toulouse Cedex 3, France (respectively)
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Mollicutes are a class of simple bacteria characterized by the lack of a bacterial cell wall and their very small genomes (580 kb to 2200 kb). This phylogenetically coherent group contains a broad range of different plant and animal pathogens making it an ideal model for understanding gene function, gene regulation and the evolution of virulence factors in other bacterial pathogens. The recent development of improved tools for manipulating mollicute genomes has transformed research in this area permitting new insights into mollicute molecular and cellular biology. An interesting fact to emerge is that, far from being a simple model of cellular life, these are complex organisms that have adapted to life in a hostile environment through a surprisingly sophisticated variety of ways.
In this book acknowledged experts critically review the most recent advances in the evolution, genetics and molecular pathogenesis of these important pathogens. Topics covered include: taxonomy; genomic mosaics; molecular genetic tools for mollicutes; identification and characterisation of virulence genes in mycoplasmas; post-translational modification of proteins; multifunctional cytoadherence factors; the glycocalyx; glycosidase activity; phase and antigenic variation in mycoplasmas; spiroplasma transmission from insect to plants; cytoskeletons organization; gliding mechanism of the Mycoplasma pneumoniae subgroup; biofilm formation by mycoplasmas; host immune responses to mycoplasmas; and emerging antimicrobial resistance in mycoplasmas of humans and animals.
An essential book for researchers working with mollicutes and recommended reading for everyone interested in bacterial genomics, bacterial pathogenesis and the evolution of bacterial virulence.
The Contentious Taxonomy of Mollicutes
Daniel R. Brown and Janet M. Bradbury
Bacterial systematics is energized by an inherent tension arising from its obligations to respect the dignity of taxa established in the past while maintaining sufficient flexibility to accommodate the advance of knowledge. The taxonomy of Mollicutes has been contentious from its inception, reflecting the challenges in cultivating the organisms axenically, early controversy over what form of life they exemplify, competing systems of nomenclature, chronological misfortune in the discovery of types, evolutionary anomalies, the sometimes enigmatic International Code of Nomenclature of Bacteria, and the usually vague species concept for prokaryotes. Their present taxonomy consequently bears acknowledged imperfections, but recent advances in genome-based systematics offer hope for future resolution of some current controversies.
It is generally assumed that the high level of host specialisation and the adoption of isolated, parasitic lifestyles that occurred during the reductive evolution of mollicutes could only result in the evolution of small, stable genomes with a high coding density. This view has been challenged recently by the analyses of an ever-increasing number of whole-genome sequences. The comparative genomics of related mycoplasma species and isolates from the same species have shown that mobile genetic elements and lateral gene transfers have produced mosaic genomes with much more plasticity than previously anticipated. This could have significant consequences on the diagnosis, virulence and taxonomy of these organisms.
Molecular Genetic Tools for Mollicutes
Joël Renaudin, Marc Breton and Christine Citti
The class Mollicutes represents a group of wall-less bacteria with small genome size and thus possesses limited metabolic capabilities. As a result of series of genome reduction and gene acquisition through horizontal gene transfer, Mollicutes have evolved a parasitic life style with the ability to colonize a wide diversity of hosts, suggesting that these organisms may use a variety of virulence mechanisms. Along with the rapid accumulation of genome sequence data, significant advances have been made in the last decade in designing new genetic tools for mollicutes. These include new selective markers, new inducible gene expression systems, and new replicative vectors for plant pathogenic spiroplasmas as well as improved Tn4001 derivatives for random mutagenesis or gene delivery, extended oriC plasmid-based strategies for new mycoplasma species for gene expression or targeted gene disruption, the production of unmarked mutations and the use of fluorescent protein genes as reporters,. Beyond the description of these new genetic tools, this review also highlights their input to the concomitant advances that have been made in understanding the intricate and complex mollicute-host interactions.
Identification and Characterisation of Virulence Genes in Mycoplasmas
Glenn F. Browning, Amir H. Noormohammadi and Philip F. Markham
The pathogenesis of most mycoplasmoses is predominantly attributable to the immunopathological response of the host to the persistent presence of these pathogens. Therefore, virulence genes in mycoplasmas are probably best defined as those that are not necessary for growth in vitro, but that are required for optimal colonisation of, persistence in or pathological effects on the host, including those genes whose primary function appears to be ensuring optimal nutrient acquisition in vivo. While the full array of virulence genes is far from being defined in any one mycoplasmal pathogen, there has been significant progress in recent years in defining the roles of adhesins, invasion, toxin production, immune evasion, immunostimulation and immunosuppression in some mycoplasmas, and in characterising some of the genes involved in these processes. In addition, it has become clear that several systems for acquisition and utilization of specific nutrients are required for optimal pathogenicity, in some cases because these nutrients contribute to the generation of toxic metabolites, while in others presumably because they facilitate persistence in a nutrient limited environment. Most recently some of the mechanisms used to regulate transcription of virulence genes have been identified, and the complexity of post-translational control of virulence factors is beginning to be revealed. These recent findings have demonstrated that mycoplasmas are far more complex pathogens than their superficially simple genomes would suggest.
Post-translational Modification of Proteins in the Mollicutes
Steven P. Djordjevic and Jessica L. Tacchi
The mycoplasmas are a genome-reduced, highly diverse group of bacteria. Although proteins constitute a significant proportion of mycoplasma membranes, genome sequence analyses reveal the presence of only a rudimentary general secretory pathway. Consequently, it is unclear how a wide array of proteins, including adherence and other virulence proteins, ABC transport proteins and key proteolytic enzymes, are translocated across their single bi-lipid membrane. Many species are host-specific and rely heavily on their hosts for the supply of essential metabolites. Nonetheless, different species employ remarkably diverse strategies to successfully colonise their respective hosts. Post-translational modifications (PTMs) of proteins profoundly influence the structure, and consequently the functions, of proteins and play fundamental roles in cellular physiology. PTMs are an integral component of the protein secretion apparatus and influence where proteins localise in cellular compartments and how they form functional complexes via interactions with other proteins. To date, post-translational modifications to mycoplasma proteins have been described in molecules that play key roles in host colonisation, metabolism and immune evasion.
Multifunctional Cytoadherence Factors
Miriam Hopfe and Birgit Henrich
In the cell-wall-less Mollicutes cytoadherence factors that enable tight contact with the host often play additional roles in colonisation, nutritional uptake and the host's immune response. Multifunctional cytoadhesins have been shown to interact with components of the extracellular matrix facilitating invasion of the host, and to hamper a successful immune response by through antigenic variation, mimicy or release of antigens into their surroundings. Cytoadhesins that condense at a tip structure in some species are also involved in gliding motility. Detailed sequence analyses have enabled the detection of six mycoplasmal cytoadhesive moonlighters that are characterised by the independence of their different functions. Heparin binding of P97 and P110 of M. hyopneumoniae was shown to be mediated by regions distinct from their cytoadhesive domains. The α-enolase of M. gallisepticum acts at different locations, intracellularly as a cytoplasmic glycolytic enzyme and surface-exposed as an ECM-binding cytoadhesin. Antigenic mimicry of P1 and P30 are unique features of these proteins in M. pneumoniae, in contrast to their homologous in other mycoplasmas. The most outstanding moonlighter described thus far is OppA of M. hominis: it mediates cytoadherence, functions as peptide-binding domain for the oligopeptide permease and is the main ecto-ATPase, the action of which leads to apoptotic death in host cells. Phosphorylation appears to be one mechanism enabling coordinated cooperation between these distinct functions in a multifunctional cytoadhesin.
The Glycocalyx of Mollicutes
James M. Daubenspeck, David S. Jordan and Kevin Dybvig
Virtually all bacteria have a glycocalyx, a sugar shell, and probably all bacterial pulmonary pathogens produce a capsule under appropriate conditions. The glycomoeities produced by these bacteria are often critical for immune evasion and survival in the animal host. The mycoplasmas are no exception. Most, if not all, mycoplasmas that are animal pathogens produce polysaccharides, glycolipids and glycoproteins. In addition, the mycoplasmas can and do adsorb glycoconjugates from their environment that are incorporated into the glycocalyx and serve to further camouflage the organism from host immunity. The adsorption of host molecules contributes to the difficulty in determining which glycoconjugates are produced by the mollicutes. The machinery for glycoconjugate synthesis in mycoplasmas is for the most part unknown. Some mycoplasmal glycosyltransferases closely resemble those of other bacteria and thus are implicated in the synthesis of glycolipids. The machinery for polysaccharide synthesis in mycoplasmas is currently obscure and potentially novel.
Glycosidase Activity in Mollicutes
Meghan May and Daniel R. Brown
Non-metabolic manipulation of carbohydrates is associated with pathogenicity in many bacteria, but was historically thought to be absent or very rare among the Mollicutes. The sequencing of numerous mycoplasma genomes has allowed the recognition of several features previously unrecognised or under-reported in mycoplasmas. As a result, glycosidase genes that can be phenotypically validated have been identified in numerous species. The characterization of these enzymes and the genes encoding them has led to a greater understanding of the ecology of the organisms and of horizontal gene transfer between Mycoplasma species. Such studies have also highlighted an area with potential for novel intervention strategies in the treatment and/or prevention of mycoplasmosis. Here we present a review of the known mycoplasmal glycosidases describing their biological characteristics, putative evolutionary origins and potential roles in pathogenicity.
Current Insights into Phase and Antigenic Variation in Mycoplasmas
Despite their small genomes and the lack of a protecting cell wall, many Mycoplasma species successfully establish chronic infections in diverse hosts, even in the presence of a specific immune response. This success is, in part, attributable to genetic switches that rapidly alter the expression, size or structure of their surface exposed proteins. These stochastic events create highly versatile and dynamic membranes, enabling these bacteria to escape the host immune response and enhance bacterial-host interactions essential for survival. This chapter provides an overview of the current knowledge of phase- and antigenic-variation in Mycoplasmas and documents on the diverse genetic mechanisms, such as reversible point mutation, slipped-strand mispairing and DNA rearrangement via site-specific or homologous recombination that lead to antigenic variation of distinct proteins in individual Mycoplasma species. The chapter contains one diagram illustrating the causes and effects of antigenic variation, three figures illustrating diverse mechanisms of phase variation and one table describing gene loci from Mycoplasma species that are affected by phase variation.
Spiroplasma Transmission from Insects to Plants: S. citri Proteins Involved in Transmission by Leafhopper Vectors
Laure Béven, Saskia Hogenhout, Fabien Labroussaa, Nathalie Arricau-Bouvery and Colette Saillard
Members of the genus Spiroplasma are motile, helical, wall-free eubacteria that are associated primarily with insects. Three spiroplasma species associated with plant disease are phloem restricted and transmitted in a persistent manner by sap-feeding leafhopper vectors. The spiroplasmas are acquired by the leafhoppers, traverse various insect tissues and then are transmitted to plants after having reached the leafhopper salivary glands. Thus, the spiroplasmas have to successfully passage different physical barriers of the gut and salivary glands. This involves specific protein-protein interactions between the spiroplasmas and insect tissues. Indeed, transmission electron microscopy studies indicated that spiroplasma invasion of gut epithelial cells occurs primarily by endocytosis at the brush border membranes and that upon invasion oval or flask-shaped spiroplasmas are inside membrane-bound vesicles. Confocal microscopy analyses of Spiroplasma citri-infected C. haematoceps salivary glands and cultured cell line (Ciha-1) experimentally infected by S. citri provided evidence that spiroplasmas associate with cell actin microfilaments. Adhesin-like proteins namely SARP1, ScARPs and SkARP are encoded on spiroplasma plasmids pBJS-O, pSci1-6 and pSKU146. Several other proteins including spiralin, ABC transporter solute binding protein and a phosphoglycerate kinase required for efficient transmission of S. citri by leafhoppers have been identified. The involvement of all these proteins in an interaction between the spiroplasma and the leafhopper cells is discussed in this chapter.
Organization of the Cytoskeletons of Diverse Mollicutes
Mitchell F. Balish
Like other organisms, mycoplasmas have cytoskeletons, which are proteinaceous, detergent-insoluble polymers that constitute important structural elements. As in other bacteria, these cytoskeletons play important roles in cell division and probably in chromosome segregation. Additionally, some groups of mycoplasmas have independently developed additional, novel cytoskeletal elements that control cell shape, polarity and movement, often with concomitant alterations to the conventional set of cytoskeletal proteins found within the bacterial domain. In this chapter the diversity, composition, organization, and assembly of the cytoskeletons of Mycoplasma pneumoniae, Mycoplasma genitalium, Mycoplasma mobile, Mycoplasma insons, Mycoplasma penetrans, Mycoplasma iowae, and Spiroplasma melliferum are discussed. Special attention is paid to the best-characterized cytoskeletal structure of mycoplasmas, the electron-dense core of the M. pneumoniae attachment organelle.
Gliding Mechanism of the Mycoplasma pneumoniae Subgroup: Implications from Studies on Mycoplasma mobile
Makoto Miyata and Daisuke Nakane
More than ten Mollicute species classified into the hominis and pneumoniae subgroups form a membrane protrusion at a cellular pole, and glide in this direction using mechanisms not seen in other bacterial genera. Our studies are unveiling the gliding mechanism of Mycoplasma mobile, the most rapidly motile species in the hominis subgroup. However, the gliding mechanism used by members of the pneumoniae subgroup are still unclear. Here, we review the gliding of members of the pneumoniae subgroup, in reference to that of M. mobile, with respect to features of their motility, their surface and cytoskeletal structures, their component proteins and their targets for binding to enable gliding. While the major features appear to be common between these two subgroups, no similarities have been found in the amino acid sequences of the component proteins involved in gliding motility.
Biofilm Formation by Mycoplasmas
Previously it has been difficult to explain how mycoplasmas manage to cause such severe and chronic infections given their paucity of virulence factors. In many other bacterial species adherence to a solid surface and biofilm formation are important steps in the initiation of disease. The possibility of environmental persistence and virulence in the host could also be explained by biofilm formation in mycoplasmas. Biofilms are sessile bacterial communities that live attached to each other and/or surfaces enclosed in a sugary exopolysaccharide matrix. Biofilm structure is highly variable and dependent on a number of factors, including the organism, the surface, the surrounding nutrient environment and the rate of flow of any aqueous interface. Biofilms are formed by the vast majority of mycoplasma species studied to date and the capacity to form biofilms is found in diverse species from all phylogenic groups of mycoplasmas. Intriguingly, mycoplasmas lack all of the known regulatory systems that are involved in biofilm formation in other bacterial species, but recent research is beginning to unravel the genetic basis of biofilm formation. The growth of biofilms in vitro and the use of biofilm model systems are also discussed.
Host Immune Responses to Mycoplasmas
Steven M. Szczepanek and Lawrence K. Silbart
The atypical characteristics of mycoplasmas are often associated with dysregulated host immune responses, which allow these pathogens to occupy an ecological niche associated with mucosal surfaces (and occasionally beyond). The lack of a cell wall and the presence of variable surface lipoproteins are paramount amongst these uncommon features, and host defense includes an intricate innate immune signaling system that includes TLRs, cytokines, and chemotactic molecules to attract, alert and activate leukocytes during infection. Mycoplasmas are adept at manipulating many of these signals to their own advantage, resulting in mutualistic relationships in some cases, or insidious chronic infections in others. Successful resolution of disease usually depends on robust humoral and cell-mediated immune responses, but such responses can take weeks to develop, thereby allowing the bacteria an opportunity to adapt to their environment and gain a foothold in colonized tissues. For this reason, successful prophylactic vaccines block initial colonization, thereby preventing the subsequent over-exuberant inflammatory and dysregulated adaptive immune responses. This chapter examines the intricate interplay between highly evolved host immune responses versus the highly adaptable mycoplasmas, with an eye towards identifying gaps in our knowledge that must be addressed in future research.
Emerging Antimicrobial Resistance in Mycoplasmas of Humans and Animals
Ken B. Waites, Inna Lysnyansky and Cécile M. Bébéar
Antimicrobial resistance has emerged in many types of bacteria and has spread worldwide, often as a result of selective pressure caused by overuse and misuse of antimicrobial agents in humans and animals. Clinically significant resistance to drugs such as tetracyclines, fluoroquinolones, and macrolides, has also developed in mycoplasmas and ureaplasmas of humans and animals, and appears to be increasing. These changes in susceptibility patterns have led to a renewed interest in development of standardized and reproducible methods for antimicrobial susceptibility testing to guide individual case management; surveillance for resistance locally, nationally, and internationally; and for evaluation of new antimicrobial agents. In vitro studies have been performed to induce resistance by stepwise selection followed by nucleic acid sequencing and analysis of the resistant microbes genetically to elucidate the molecular mechanisms involved. Clinical isolates proven to be resistant to various drugs phenotypically have also been characterized genetically and compared with mutants selected in vitro to clarify further the resistance mechanisms that are operative in a natural setting. In many instances, the same mechanisms have been shown to occur naturally and in vitro. In this chapter we have summarized antimicrobial agents useful for treatment of mycoplasma and ureaplasma infections of humans and animals and the current trends in development of antimicrobial resistance in these organisms. Evidence for the molecular basis of antimicrobial resistance is also discussed along with descriptions of methods for determination of antimicrobial susceptibilities.
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(EAN: 9781908230300 Subjects: [microbiology] [bacteriology] [medical microbiology] [molecular microbiology] [genomics] )