User:Chhandama/sandbox

Introduction

Flagella are motility filaments in a wide range of cells, from bacteria to those of mammals. However, the different domains of life, from bacteria to archaea to eukaryotes, do not share structural or functional identities, indicating independent evolution of the organ in individual domains. Once thought to be a symbiotic product of other free-living cells with parent (host) cell during the early stages of symbiogenesis, most likely from the filamentous spirochaete-like prokaryotes, it is now more convincingly demonstrated that they arose originally from an already formed microtubular structures that initially worked in cytoplasmic motility such as intracellular transport. In fact, the flagella, if not principally, could have been a major distinctive feature of each type of cells across the different life forms, and contributing to much more than just locomotory machinery. In each group of life domain do we see additional structural and functional features, while the fundamental microtubules are still maintained.

It may be boldly argued that among eukaryotes, the flagellum is most well developed in terms of structural and functional complexity in the protozoan belonging to the phylum, specifically in the species of Angonomas, Crithidia, Euglena, Herpetomonas, Leishmania, Peranema, Phytomonas and Trypanosoma, all related through the same phylum Euglenozoa. In contrast to any other flagellated organism or cell, they are characterised by an accessory filament called paraflagellar rod (PFR), or sometimes called paraxial rod. The complex organisation of the microtubules and the PFR proteins contribute to advanced motility characters, as well as enhancing the pathogentic capacities of the parasitic species. Trypanosoma brucei is outstanding among the PFR-associated protists in that it exclusively lead a life of permanent flagellation; its developmental stages in tsetse fly (the epimastigote) having the flagellum originating form the anterior end, and in mammals (the trypomastigote) in which the flagellum arises from the posterior region, are continuously flagellated.

The PFR, as a unique structure, has been one of the principle focus of research in the recent past. The structural organisations and functions of the PFR are fundamentally similar in different euglenid and kinetoplastid species, but are highly variable in size. A PFR is typically a three-layered mesh of over 40 different proteins that form a trilaminar lattice-like filament running alongside the microtubule. Euglenozoans exhibit unique flagellar movement during locomotion with characteristic non-planar helical motion unlike the flagella of other animals. The PFR helps in maintaining the structural integrity of the flagellum during such movements by imparting an elastic resistance during the undulation of the axoneme. It also serves as a conduit for Ca2+ and cAMP signalling pathway inside the flagellar complex, thereby controlling the mechanics of flagellar motion. It further plays a critical role in the parasite-host interaction that it may provide a target site for antiparasitic drugs.

Historical context

The flagellar composition and functional aspects, thereby the biological implications, are most extensively studied, and thus, best understood from the trypanosomes, species and variants of Trypanosoma brucei. As a matter of historical fact, eukaryotic flagella became the first best recognised in these parasites. When David Bruce, serving in the British Royal Army Medical Corps, was deployed to Zululand (now part of South Africa) to investigate the case of sleeping sickness (locally referred to as nagana or N'gana, a Zulu word for powerless or useless, for the obvious symptoms of the infection), he discovered the unique identity of the protozoan from any other known species as flagellated blood parasites, a haematozoon. As he reported in 1895:

At this point I think it will be convenient to give a definite description of the parasite discovered by me in the blood of animals affected by this disease, and to bring forward my reasons for considering it to be the proximate exciting cause of the disease. For the present I shall call it the Haematozoon or Blood Parasite of Fly disease, although in all probability on further knowledge it will be found to be identical with the haematozoon of Surra, which is called Trypanosoma Evansi or at least a species belonging to that genus... [The farsightedness of Bruce became obvious as T. evansi is considered most likely a subspecies of T. brucei] They appear to be about a quarter of the diameter of a Red Blood Corpuscle in thickness, and 2 or 3 times the diameter of a corpuscle in length. They are pointed or somewhat blunt at one end, and the other extremity is seen to be prolonged into a very fine lash, which is in constant whip-like motion. Running along the cylindrical body between the two extremities can be seen a transparent delicate longitudinal membrane or fin [later named undulating membrane] which is also constantly in wave-like motion.

The terms lash and whip are the literal meaning of the Latin word flagellum or flagrum, which was introduced as a name for the filamentous locomotory organ in the early 19th century. Bruce's discovery and description had since piqued research attention from various corners, microbiology, pathology, cell biology, molecular biology, genetics and evolution. However for understanding the finer details, micro-organ in microorganisms required extraordinary microscopy. That crux of challenge was to be solved only by the development and application of electron microscopy in the mid-20th century. In 1950, Albrecht Kleinschmidt and Ernst Kinder provided a pioneering electron microscopic analysis of the flagella of rat trypanosomes. It was in the next year that F.C. Kraneveld, A. L. Houwink and H. J. W. Keidel furnished the first ultrastructural description from T. evansi. However, their microscopic study did not give much of the structural details as the samples were viewed only in the longitudinal positions.

In 1956, Patricia C. H. Chang at the Johns Hopkins University, US, first made a fine cross section of the Leishmania donovani. Her images, though of poor visibility, showed at least that the flagellum was not a simple circular or cylindrical filament as in other organisms, there was an extra-axonemal component; she described: "The dense ring with the hollow center (axoneme) is still present; however, in addition to this structure, there is another less dense granular ring partially surrounding the axoneme." Of course, she did not identify the additional filament. In 1958, C.K. Pyne from the Institute of Nuclear Physics in Calcutta (Kolkata), India, published one of the best microscopical description with fine transverse sections of Leishmania donovani. His paper clearly revealed an unusual structural component beside the axoneme within the flagellum – the presence of an oval-shaped dark blob adjacent to the axoneme. His microscopic image had still a poor resolution. Perplexed by this structure, he passing described: "The nature of the body seen besides the fibrils is not clear." The first clear picture and analysis of the elusive structure was given by L.E. Roth from the Argonne National Laboratory, Illinois, US, in Peranema trichophorum, a free-living protist that bears a pair of flagella. Roth gave a vivid description:

If the more bulbous portion of the reservoir is cross-sectioned, both flagella are usually visible and may also be cross- sectioned so that the 11 fibrils typical of cilia and flagella are observed. However, an intraflagellar structure of a diameter greater than the fibril bundle is also present within the flagellar membrane and forms a long, tapering rod which is still present in more distal sections though in a reduced diameter.

Roth introduced the name "intraflagellar material" which would be later variously known as paraflagellar rod or paraxial rod. Ironically, the works of Pyne and Roth remain largely forgotten, and the first description of PFR is almost always credited to Keith Vickerman,  who reported his description in 1959 on T. brucei. Bastin et al. (1996) were the most explicit in their documentary article, saying: "The paraflagellar rod (PFR) was first identified in trypanosomes by Keith Vickerman in 1962." Such is the perpetual misinformation. Vickerman was no better in his descriptive analysis than Pyne and Roth, as he could merely identify the PFR as "accessory filament."

The flagellar complex
In all organisms and individual cells possessing flagella, the only internal component is the axoneme, which in turn is composed of microtubules and dyneins. Only in the euglenozoans, such as kinetoplastids (Crithidia, Herpetomonas, Leishmania, Phytomonas and Trypanosoma species) and euglenid (Euglena species), are the flagellum composed of two distinct parts, the axoneme and PFR.

In the trypomastigote and epimastigote forms of kinetoplastids, the flagellum arises from the posterior or middle portion of the main body so that the flagellar membrane and the cell membrane forms a continuous structure known as undulating membrane that creates wavy motions during locomotion. The adhesion of the flagellar membrane to the body surface along the undulating membrane is mediated by the flagellum attachment zone, which is a complex of cytoskeletal filaments. The PFR is attached to the inner surface of the undulating membrane on one side and to the flagellum attachment zone on the other.

As in a typical eukaryote, the axoneme of euglenozoans contains nine doublet (each doublet is a pair of two, A and B tubules) microtubules arranged in a circle that surrounds two central singlet (unpaired) microtubules giving a structural configuration of (9+2) filament. This is in contrast with prokaryotes, where there are no central filaments, giving the flagellum a characteristic (9+0) filament. Again unlike in prokaryotes in which the flagella are a rotor machineries, the beat of the eukaryotic axoneme is powered by the sliding of the doublet microtubules against each other, the sliding of which is generated by the ATPase motor proteins, the dyneins, that are attached to the A tubule. The relative sliding and twisting are maintained by the radial spokes, a complex of over 20 proteins, that link the doublet microtubules to the central pair.

The flagellum is attached to the base of the cell membrane where the body forms an invagination called flagellar pocket. The flagellum emerges out of this pocket and maintains attachment with the cell membrane (forming the undulating membrane) along the length of its body, until it reached the anterior tip from where it is a free filament. It is the only site of molecular exchange in and out of the body. Within the flagellar pocket the flagellum has variable structural organisation. At the very base, the microtubules take the form of (9+0) triplet microtubules, as seen in centrioles of normal eukaryotic cells, especially those about to divide. This is the basal body, where the microtubules originate, and embedded in the cell membrane. The attachment of the basal body to the cell body mediated by independent microtubules that lines the internal side of the cell membrane via a region known as the flagellum attachment zone (FAZ). The FAZ is specifically composed of four microtubules, the microtubule quartet. On top of the basal body toward and within the flagellar pocket is another configuration, a (9+0) doublet filament. This filament continues up to the flagellar pocket collar, the boundary to the external environment. From that point, the axoneme continues as a (9+2) doublet filament, running along side the PFR.

Molecular architecture
As in typical eukaryotes, the flagella in trypanosomes bear the canonical (9+2) axoneme consisting of nine circular doublet microtubules enclosing two central singlet microtubules. The interlinked microtubules together form the axoneme, the functional structure of the flagellum. A PFR appears under a transmission electron microscope as a three-layered lattice-like filament that runs along the entire length of the flagella, except at the base called flagellar pocket. The microtubules and the PFR are together enclosed entirely by a flagellar membrane. It is present in all the life cycle stages, including trypomastigote, epimastigote and promastigote, of kinetoplastids but not in the amastigote. In an amastigote stage, such as found in Leishmania species and Trypanosoma cruzi, there is no PFR and external flagellum, but only a highly reduced axoneme, or a residual flagellum, that is confined to the flagellar pocket inside the cytoplasm. Thus, this kinetoplastid stage is also referred to as "aflagellate amastigote form" or the individuals as "aflagellated amastigotes."

The PFR has a molecular architecture that is almost similar to intermediate filaments found in invertebrates and vertebrates as one of the cytoskeletons; the others being microtubules and microfilaments. Like intermediate filament, it has high solubility in trypsin, resistance to detergent treatment, similar amino acid composition, and a helical orientation. However, its association with flagellum and specific configuration indicate that it is not a typical cytoskeleton. The general overall thickness of the PFR is 150 nm throughout the entire length. It has three well-defined regions relative to the positions within the axoneme, namely the proximal, the intermediate and the distal zones (or domains). The proximal domain is highly conserved in that the structure is basically same across different species. However, the intermediate and the distal domains exhibit specific organisations of the thick and thin filaments giving rise to variations in the overall size in different species.

The fine structural organisation of the proximal and distal zones have common features. They are both dense plates composed of thin filaments measuring 7–10 nm in width and thick double filaments of 25 nm wide. The proximal domain lies closest to the microtubule and form a peptide link with the outer doublets using four to seven electron dense filaments, while the distal zones are farthest away from the axonome and form the thickest portion. The intermediate domain consists only of thin filaments of 5 nm wide that connect the proximal and distal domains on either sides. The proximal zone is connected to the axoneme by a set of proteins called PFR-axoneme connectors (PACs).

In T. brucei, where the PFR is most extensively studied, the filaments are stacked in bundles. The bundles, called "scissors densities," are arranged 54 nm from each other and oriented at 45° to the axoneme axis. They are connected by thin filaments called "wire densities." Such arrangement is essential for structural integrity while being flexible. The repeating densities give rise to an appearance of a comb teeth along the longitudinal axis. There can be ~370 scissor density planes with each plane containing ~27 pairs of scissors for a 20-µm-long PFR.

Functions
In the flagellum, the axoneme is the main locomotory structure, while PFR performs diverse functional roles as an accessory filament. The PFR helps in maintaining the structural integrity of the flagellum during movements by imparting an elastic resistance during the undulation of the axoneme. It acts as a mechanical spring by absorbing, storing and relaying the forces generated by axonemal beating. Therefore, it adds to the efficiency of the flagellar movement which is crucial in euglenozoans as they spend much of their lifetime moving in the host's body fluids. Particularly in T. brucei, the only species which spends its entire lifecycle in persistent motion, except during cell divisions for reproduction, an extra efficient flagellar movement is required. Indirect evidence indicates that the mechanochemical signals of the flagellar motility originate in the PFR, from which the signals are relayed to the dynein arms of the axoneme.

However, the euglenozoan axoneme is functionally different from those of other eukaryotes in that it generates motion as a series of beats consisting of bihelical waves, and each beat starts from the terminal tip and ends at the base. The bihelical waves are alternating left-handed and right-handed forces and separated by relatively relaxed states or kinks after each wave along the axoneme. This creates a drill-like motion on the entire length of the flagellum. This is the very origin of the name trypanosome, derived form the greek words τρυπανον (trypanon or trupanon), which means "borer" or "auger", referring to the corkscrew-like movement. As a result of such motion, the body is moved in the direction of the tip of the flagellum. This is unusual in the sense that other flagellated organisms or cells, say bacteria and spematozoa, are propelled by the flagella from the posterior end. In this way, the trypanosome flagellum is better described as a hydrodynamic dragging filament, that pulls the entire cell body towards the very tip of the flagellum.

The flagellum of trypanosomes are known to confer the pathogenicity of the parasites in direct and indirect ways. Indirectly, it maintains and controls the reproductive process of the epimastigotes, while ensuring a population of infective trypomastigotes inside the salivary glands of the host tsetse fly. It also directly contributes to the pathogenesis of the infection at cellular levels. This pathogenicity is in turn achieved by at least two major biological processes: immune evasion by transport and elimination of antibodies and secretion of virulence factors.

Functional role in infectivity

Although the nuclear and kinetoplast genomes dictate the central process of cell division leading to a complete reproduction (by binary fission) in trypanosomes, the flagellum holds a key to successful division. The involvement of flagellum, especially in the epimastigote forms that produce the mammal-infective trypomastigotes, is critical in maintaining two populations of trypanosomes in the insect host. Flagellum promotes asymmetrical cell divisions that give rise to one of the most complex binary fissions. The peculiar reproduction takes place in two successive stages and locations. First in the proventriculus of the insect, each epimastigote divides into two forms, long and short epimastigotes. The long individuals appear to lack flagellar motility as they cannot migrate to other places and simply die off.

The surviving short individuals then relocate to the salavary glands where they undergo another round of cell division. Similar asymmetric mitosis produces two different daughter cells from the mother epimastigote. One daughter is an epimastigote that remains non-infective and the other is a trypomastigote which undergoes transformation into the infective forms. In this way, an unequal binary fission, promoted by flagellar division continuously creates the formation of non-infective epimastigotes that can continue other rounds of cell division, and another population of infective trypomastigotes. The disparate daughter cells in shape and length are due to the unequal growth of the flagella. As the process of reproduction is executed, the daughter epimastigotes and trypomastigotes are buried into the epithelial microvilli on the walls of the salivary glands using their flagella. As a matter of fact, it is during this specific attachment that the VSGs are produced. Thus, the trypanosome flagellum is not only a motility filament, but an attachment organ as well, and is necessary for maintaing the infective forms in the insect slaivary glands.

Function in virulence and infection

As the flagellar complex is the only site of molecular exchange (endocytosis and exocytosis) for the parasites with its environment, it is the critical site in host-parasite interaction. Although the molecular details are yet to be elucidated, it in now obvious that the flagellar membrane secretes proteins that are essential to triggering the virulence mechanisms in trypanosomes. A varied group of proteins are involved as virulence factors, which are produced as membranous nanotubes from the flagellar membrane. The membrane then forms transport vesicles carrying the proteins out of the parasite into the host's blood stream. For example, the key molecules in the pathogenic invasion and immune evasion are due to a cohort of variant surface glycoproteins (VSGs) produced on the trypanosome cell surface. Inside the mammalian blood streams, the production of these VSGs are stimulated by glycosylphosphatidylinositol-phospholipase C (GPI-PLC) that is concentrated in the flagellar membrane. This GPI-PLC is an enzyme that is not the main virulence factor but is apparently required for the full virulence during infection. It is also essential for shedding the VSGs from the trypanosomes in mammalian blood while transforming to insect-infective forms, thus, indicating that they are required for normal turn over of VSG and maintaining the infective properties of the parasites.

Among other flagellar molecules that contribute to the virulence are calflagins and metacaspase 4 (MCA4). Calflagins are Ca2+-binding proteins that are involved in antibody clearance in bloodstream parasites. MCA4 is a pseudopeptidase lacking any detectable peptidase activity, but crucial during blood infection. Although the details of virulence are incompletely known, MCA4-null mutants are characterised to have reduced virulence in experimental conditions. Yet another protein ESAG4 (expression site associated gene 4) is present in the bloodstream form. Identified as member of a large family of adenylyl cyclases that catalyse the conversion of ATP to cAMP, the enzyme i partly essential to maintain the full virulence of the parasites. T. brucei genome codes for five specific phosphodiesterases, two (TbrPDEBl and TbrPDEB2) of which are essential in virulence. TbrPDEB1 is present in the flagellar membrane, whereas TbrPDEB2 is distributed both in the flagellum and cytoplasm.

The flagellar complex is also key to direct immune evasion during infection of mammals. The immune evasive process is a notable mechanical process. The initial immune response from the host in the form of antibodies (immunoglobulins, IgG and IgM) are readily captured by VSGs in the blood circulation. The Ig-VSG complexes are transported into the parasite body for proteolytic destruction, thereby abrogating the critical immune signals. The Ig-VSG complexes are not taken up by ordinary endocytosis, but preceded by a flagellar motion. The actively swimming trypanosomes literally sweep the Ig-VSG complexes adhering on any location of the cell surface using the flagella. The flagella then push the molecules towards the flagellar pocket, where they can be taken up into the cytoplasm for ultimate degradation. The host mammal is thereby prevented from recognising the infection.The whole scenario presents an insight into the complexity of trypanosome flagellar forms and capabilities, and is an evidence that much more is to be unveiled.