Leishmania

Leishmania is a genus of trypanosomatid protozoa, and is the parasite responsible for the disease leishmaniasis. It is spread through sandflies of the genus Phlebotomus in the Old World, and of the genus Lutzomyia in the New World. Their primary hosts are vertebrates; Leishmania commonly infects hyraxes, canids, rodents, and humans. Leishmania currently affects 12 million people in 98 countries.

The parasite was named in 1903 after the Scottish pathologist William Boog Leishman.

Origin
The origins of Leishmania are unclear. One theory proposes an African origin, with migration to the Americas. Another proposes migration from the Americas to the Old World about 15 million years ago, across the Bering Strait land bridge. Another proposes a palearctic origin. Such migrations would entail migration of vector and reservoir or successive adaptations along the way. A more recent migration is that of L. infantum from Mediterranean countries to Latin America (there named L. chagasi), since European colonization of the New World, where the parasites picked up its current New World vectors in their respective ecologies. This is the cause of the epidemics now evident. One recent New World epidemic concerns foxhounds in the USA.

Biochemistry and cell biology
The biochemistry and cell biology of Leishmania is similar to that of other kinetoplastids. They share the same main morphological features; a single flagellum which has an invagination, the flagellar pocket, at its base, a kinetoplast which is found in the single mitochondrion and a sub-pelicular array of microtubles which make up the main part of the cytoskeleton.

Lipophosphoglycan coat
Leishmania possess a lipophosphoglycan coat over the outside of the Leishmania cell. Lipophosphoglycan is a trigger for Toll-like receptor 2, a signalling receptor involved in triggering an innate immune response in mammals.

Structure
The precise structure of lipophosphoglycan varies depending on the species and life cycle stage of the parasite. The glycan component is particularly variable and different lipophosphoglycan variants can be used as a molecular marker for different life cycle stages. Lectins, a group of plant proteins which bind different glycans, are often used to detect these lipophosphoglycan variants. For example peanut agglutinin binds a particular lipophosphoglycan found on the surface of the infective form of Leishmania major.

Function
Lipophosphoglycan is used by the parasite to promote its survival in the host and the mechanisms by which the parasite does this center around modulating the immune response of the host. This is vital as the Leishmania parasites live within macrophages and need to prevent the macrophage from killing them. Lipophosphoglycan has a role in resisting the complement system, inhibiting the oxidative burst response, inducing an inflammation response and preventing natural killer T cells recognising that the macrophage is infected with the Leishmania parasite.

Pathophysiology
Leishmania cells have two morphological forms: promastigote (with an anterior flagellum) in the insect host, and amastigote (without flagella) in the vertebrate host. Infections are regarded as cutaneous, mucocutaneous, or visceral.

Neutrophil granulocytes – the Trojan horses for Leishmania parasites
The strategy of the "Trojan horse" as a mechanism of pathogenicity of intracellular microorganisms is to avoid the immune system and its memory function cleverly with phagocytosis of infected and apoptotic neutrophils by macrophages and employing the non-danger surface signals of apoptotic cells.

Transmitted by the sandfly, the protozoan parasites of the genus Leishmania major may switch the strategy of the first immune defense from eating/inflammation/killing to eating/no inflammation/no killing of their host phagocyte' and corrupt it for their own benefit. They use the willingly phagocytosing polymorphonuclear neutrophil granulocytes (PMN) rigorously as a tricky hideout, where they proliferate unrecognized from the immune system and enter the long-lived macrophages to establish a “hidden” infection.

Uptake and survival


By a microbial infection PMN move out from the bloodstream and through the vessels’ endothelial layer, to the site of the infected tissue (dermal tissue after fly bite). They immediately start their business there as the first immune response and phagocytize the invader because of the foreign and activating surfaces. In that processes an inflammation emerges. Activated PMN secrete chemokines,  IL-8 particularly, to attract further granulocytes and stimulate them to phagocytosis. Furthermore Leishmania major increases the secretion of IL-8 by PMN. In the parasites case, that may not sound reasonable at first. We can observe this mechanism on other obligate intracellular parasites, too. For microbes like these, there are several ways to survive inside cells. Surprisingly, the co-injection of apoptotic and viable pathogens causes by far a more fulminate course of disease than injection of only viable parasites. Exposing on the surface of dead parasites the anti-inflammatory signal phosphatidylserine, usually found on apoptotic cells, Leishmania major switches off the oxidative burst, so killing and degradation of the co-injected viable pathogen is not achieved.

In the case of Leishmania, progeny are not generated in PMN, but in this way they can survive and persist untangled in the primary site of infection. The promastigote forms also release LCF (Leishmania chemotactic factor) to actively recruit neutrophils, but not other leukocytes, for instance monocytes or NK cells. In addition to that, the production of interferon gamma (IFNγ)-inducible protein 10 (IP10) by PMN is blocked in attendance of  Leishmania, what involves the shut down of inflammatory and protective immune response by NK and Th1 cell  recruitment. The pathogens stay viable during phagocytosis since their primary hosts, the PMN, expose apoptotic cell associated molecular pattern (ACAMP) signaling “no pathogen.”

Persistency and attraction
The lifespan of neutrophil granulocytes is quite short. They circulate in bloodstream for about 6 or 10 hours after leaving bone marrow, whereupon they undergo spontaneous apoptosis. Microbial pathogens have been reported to influence cellular apoptosis by different strategies. Obviously because of the inhibition of caspase3-activation Leishmania major can induce the delay of neutrophils apoptosis and extend their lifespan for at least 2–3 days. The fact of extended lifespan is very beneficial for the development of infection because the final host cells for these parasites are macrophages, which normally migrate to the sites of infection within 2 or 3 days. The pathogens are not dronish; instead they take over the command at the primary site of infection. They induce the production by PMN of the chemokines MIP-1α and MIP-1β (macrophage inflammatory protein) to recruit macrophages.

Silent phagocytosis Theory
To save the integrity of the surrounding tissue from the toxic cell components and proteolytic enzymes contained in neutrophils, the apoptotic PMN are silently cleared by macrophages. Dying PMN expose the "eat me"-signal phosphatidylserine which is transferred to the outer leaflet of the plasma membrane during apoptosis. By reason of delayed apoptosis the parasites that persist in PMN are taken up into macrophages, employing an absolutely physiological and non-phlogistic process. The strategy of this "silent phagocytosis" has following advantage for the parasite:


 * Taking up apoptotic cells silences macrophage killing activity leading to a survival of the pathogens.
 * Pathogens inside of PMN have no direct contact to the macrophage surface receptors, because they can not see the parasite inside the apoptotic cell. So the activation of the phagocyte for immune activation does not occur.

However studies have shown this is unlikely the case as the pathogens are seen to leave apoptopic cells and there is no eveidence of macrophage uptake via this method.

Treatment
Pentavalent antimonial compounds such as sodium stibogluconate and meglumine antimoniate are the traditional treatments for leishmaniasis. Among all of the computationally screened compounds, pentamidine, 1,3-dinitroadamantane, acyclovir and analogs of acyclovir had higher binding affinities than the real substrate (guanosine monophosphate). Amino acids of HGPRT that are frequently involved in the binding of these compounds are Lys 66, Asp 74, Arg 77, Asp 81, Val 88, Tyr 182, Arg 192 and Arg 194. It is predicted that patients suffering from both HIV and visceral leishmaniasis (VL) may benefit if they are treated with acyclovir or pentamidine in conjunction with first-line antileishmanial therapies such as miltefosine and AmBisome.Ansari et al. Resistance to the antimonials is prevalent in some parts of the world, and the most common alternative is amphotericin B (see leishmaniasis for other treatment options). Paromomycin is an inexpensive alternative with fewer side effects than amphotericin that The Institute for OneWorld Health has funded for production as an orphan drug for use in treatment of leishmaniasis, starting in India.

New research in the Netherlands on a cancer drug called Miltefosine has shown promising results in treating Leishmaniasis. Miltefosine is the first effective oral treatment for leishmaniasis and is currently undergoing human trials. On its own it has an 88.2% success rate in curing Leishmania but when paired with antimonials the success rate jumps to near 100%.

Common adverse effects of the miltefosine treatment are nausea, abdominal discomfort and temporary diminution of ejaculate volume, but few patients discontinue treatment as a result of these adverse effects. Miltefosine is embryotoxic and teratogenic, prohibiting use during pregnancy and, because of its long residence time, requires effective contraception up to at least 5 months after treatment.

Molecular biology
An important aspect of the Leishmania protozoan is its glycoconjugate layer of lipophosphoglycan (LPG). This is held together with a phosphoinositide membrane anchor, and has a tripartite structure consisting of a lipid domain, a neutral hexasaccharide, and a phosphorylated galactose-mannose, with a termination in a neutral cap. Not only do these parasites develop post-phlebotomus digestion but, it is thought to be essential to oxidative bursts, thus allowing passage for infection. Characteristics of intracellular digestion include an endosome fusing with a lysosome, releasing acid hydrolases which degrade DNA, RNA, proteins and carbohydrates.

Genomics


The genomes of three Leishmania species (L. major, L. infantum and L. braziliensis) have been sequenced, revealing more than 8300 protein-coding and 900 RNA genes. Almost 40% of protein-coding genes fall into 662 families containing between two and 500 members. Most of the smaller gene families are tandem arrays of one to three genes, while the larger gene families are often dispersed in tandem arrays at different loci throughout the genome. Each of the 35 or 36 chromosomes are organized into a small number of gene clusters of tens-to-hundreds of genes on the same DNA strand. These clusters can be organized in head-to-head (divergent) or tail-to-tail (convergent) fashion, with the latter often separated by tRNA, rRNA and/or snRNA genes. Transcription of protein-coding genes initiates bi-directionally in the divergent strand-switch regions between gene clusters and extends polycistronically through each gene cluster before terminating in the strand-switch region separating convergent clusters. Leishmania telomeres are usually relatively small, consisting of a few different types of repeat sequence. Evidence can be found for recombination between several different groups of telomeres. The L. major and L. infantum genomes contain only ~50 copies of inactive degenerated Ingi/L1Tc-related elements (DIREs), while L. braziliensis also contains several telomere-associated transposable elements (TATEs) and spliced leader-associated (SLACs) retroelements. The Leishmania genomes share a conserved core proteome of ~6200 genes with the related trypanosomatids Trypanosoma brucei and Trypanosoma cruzi, but there are ~1000 Leishmania-specific genes (LSGs), which are mostly randomly distributed throughout the genome. There are relatively few (~200) species-specific differences in gene content between the three sequenced Leishmania genomes, but ~8% of the genes appear to be evolving at different rates between the three species, indicative of different selective pressures that could be related to disease pathology. About 65% of protein-coding genes currently lack functional assignment.

Leishmania species produce several different heat shock proteins. These incluse Hsp83, a homolog of Hsp90. A regulatory element in the 3' UTR of Hsp83 controls translation of Hsp83 in a temperature-sensitive manner. This regions forms a stable RNA structure which melts at higher temperatures.

Leishmania as component of CVBD
Other microorganism-based diseases caused by ectoparasites include Bartonella, Borrelia,  Babesia, Dirofilaria, Ehrlichia, and Anaplasma.

Literature

 * Zandbergen et al. "Leishmania disease development depends on the presence of apoptotic promastigotes in the virulent inoculum", PNAS, Sept. 2006 (PDF)