Chaoqun Yao

The major surface protease (MSP or GP63) of Leishmania sp. Biosynthesis, regulation of expression, and function

Leishmania sp. are digenetic protozoa that cause an estimated 1.5-2 million new cases of leishmaniasis per year worldwide. Among the molecular factors that contribute to Leishmania sp. virulence and pathogenesis is the major surface protease, alternately called MSP, GP63, leishmanolysin, EC3.4.24.36, and PSP, which is the most abundant surface protein of leishmania promastigotes. Recent studies using gene knockout, antisense RNA and overexpression mutants have demonstrated a role for MSP in resistance of promastigotes to complement-mediated lysis and either a direct or indirect role in receptor-mediated uptake of leishmania. The MSP gene clusters in different Leishmania sp. include multiple distinct MSPs that tend to fall into three classes, which can be distinguished by their sequences and by their differential expression in parasite life stages. Regulated expression of MSP class gene products during the parasite life cycle occurs at several levels involving both mRNA and protein metabolism. In this review we summarize advances in MSP research over the past decade, including organization of the gene families, crystal structure of the protein, regulation of mRNA and protein expression, biosynthesis and possible functions. The MSPs exquisitely demonstrate the multiple levels of post-transcriptional gene regulation that occur in Leishmania sp. and other trypanosomatid protozoa.

Biosynthesis of the major surface protease GP63 of Leishmania chagasi

The protozoan Leishmania chagasi expresses a surface metalloprotease, GP63, whose abundance increases 14-fold as parasites grow from logarithmic to stationary phase. L. chagasi GP63 is encoded by three classes of MSP genes that are differentially expressed during parasite growth. Using metabolic labeling and immunoprecipitation, we found L. chagasi GP63 first appeared as a 66-kDa band that was replaced by a 63-kDa protein. This pattern also occurred in transfected L. donovani harboring detectable products of only one MSP gene, suggesting a precursor-product relationship. The half-life of GP63 increased from 29 h in logarithmic phase to >72 h in stationary phase promastigotes. GP63 loss from the cell was complemented by the appearance of a 63-kDa GP63 in extracellular medium in both membrane-associated and -free forms. Calculations suggested that the long and lengthening T(1/2) of cell-associated GP63 accounts in part for its progressive accumulation in the cell during promastigote growth. The current findings add yet another level of complexity to post-transcriptionally regulated expression of an abundant surface molecule in a trypanosomatid protozoan.

Internal and Surface-Localized Major Surface Proteases of Leishmania spp. and Their Differential Release from Promastigotes

ABSTRACT Major surface protease (MSP), also called GP63, is a virulence factor of Leishmania spp. protozoa. There are three pools of MSP, located either internally within the parasite, anchored to the surface membrane, or released into the extracellular environment. The regulation and biological functions of these MSP pools are unknown. We investigated here the trafficking and extrusion of surface versus internal MSPs. Virulent Leishmania chagasi undergo a growth-associated lengthening in the t1/2 of surface-localized MSP, but this did not occur in the attenuated L5 strain. The release of surface-localized MSP was enhanced in a dose-dependent manner by MβCD, which chelates membrane cholesterol-ergosterol. Furthermore, incubation of promastigotes at 37°C with Matrigel matrix, a soluble basement membrane extract of Engelbreth-Holm-Swarm tumor cells, stimulated the release of internal MSP but not of surface-located MSP. Taken together, these data indicate that MSP subpopulations in distinct cellular locations are released from the parasite under different environmental conditions. We hypothesize that the internal MSP with its lengthy t1/2 does not serve as a pool for promastigote surface MSP in the sand fly vector but that it instead functions as an MSP pool ready for quick release upon inoculation of metacyclic promastigotes into mammals. We present a model in which these different MSP pools are released under distinct life cycle-specific conditions.

Proteomic examination of Leishmania chagasi plasma membrane proteins: Contrast between avirulent and virulent (metacyclic) parasite forms

Purpose: About two million new cases of leishmaniasis with 50 000 associated deaths occur worldwide each year. Promastigotes of the causative Leishmania spp. develop from the procyclic stage to the highly virulent metacyclic stage within the sand fly vector. We hypothesized that proteins important for promastigote virulence might be uniquely represented in the plasma membrane of metacyclic, but not procyclic, promastigotes.

Dynamics of sterol synthesis during development of Leishmania spp. parasites to their virulent form

BackgroundThe Leishmania spp. protozoa, the causative agents of the “neglected” tropical disease leishmaniasis, are transmitted to mammals by sand fly vectors. Within the sand fly, parasites transform from amastigotes to procyclic promastigotes, followed by development of virulent (metacyclic) promastigote forms. The latter are infectious to mammalian hosts. Biochemical components localized in the parasite plasma membrane such as proteins and sterols play a pivotal role in Leishmania pathogenesis. Leishmania spp. lack the enzymes for cholesterol synthesis, and the dynamics of sterol acquisition and biosynthesis in parasite developmental stages are not understood. We hypothesized that dynamic changes in sterol composition during metacyclogenesis contribute to the virulence of metacyclic promastigotes.MethodsSterols were extracted from logarithmic phase or metacyclic promastigotes grown in liquid culture with or without cholesterol, and analyzed qualitatively and quantitatively by gas chromatograph-mass spectrometry (GC-MS). TriTrypDB was searched for identification of genes involved in Leishmania sterol biosynthetic pathways.ResultsIn total nine sterols were identified. There were dynamic changes in sterols during promastigote metacyclogenesis. Cholesterol in the culture medium affected sterol composition in different parasite stages. There were qualitative and relative quantitative differences between the sterol content of virulent versus avirulent parasite strains. A tentative sterol biosynthetic pathway in Leishmania spp. promastigotes was identified.ConclusionsSignificant differences in sterol composition were observed between promastigote stages, and between parasites exposed to different extracellular cholesterol in the environment. These data lay the foundation for further investigating the role of sterols in the pathogenesis of Leishmania spp. infections.

Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

ABSTRACT The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. MβCD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, MβCD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of MβCD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins.

Aberrant and accidental trichomonad flagellate infections: rare or underdiagnosed?

The clinical significance and frequency of human infections with trichomonad flagellates in aberrant locations (locations other than the predilection sites) and accidental infections (infections with non-human trichomonads) are unclear. The total number of case reports in the literature is low, with the identification of the infection largely the outcome of investigations for the cause of clinical signs. At least seven species have been identified in either aberrant or accidental infections, with these infections occurring worldwide. Results of prospective and retrospective surveys of adults with respiratory disease suggest that aberrant and accidental trichomonad infections are less rare than the number of case reports suggest, with Trichomonas tenax being the most frequent. Surveys of neonates and infants suggest that aberrant Trichomonas vaginalis infections can be higher and more serious than suggested by the few case reports. The density and location of infection might be as important, if not more so, than species in determining pathogenicity. Molecular diagnostic methods including DNA sequencing can be used to better diagnose infections and enable identification to assist in determining the clinical significance of different species. Raising awareness of these infections among clinicians and adding them to their differential lists are strongly suggested.

Extracellular release of virulence factor major surface protease via exosomes in Leishmania infantum promastigotes

BackgroundThe Leishmania spp. protozoa are introduced into humans through a sand fly blood meal, depositing the infectious metacyclic promastigote form of the parasite into human skin. Parasites enter a variety of host cells, although a majority are found in macrophages where they replicate intracellularly during chronic leishmaniasis. Symptomatic leishmaniasis causes considerable human morbidity in endemic regions. The Leishmania spp. evade host microbicidal mechanisms partially through virulence-associated proteins such as the major surface protease (MSP or GP63), to inactivate immune factors in the host environment. MSP is a metalloprotease encoded by a tandem array of genes belonging to three msp gene classes, whose mRNAs are differentially expressed in different life stages of the parasite. Like other cells, Leishmania spp. release small membrane-bound vesicles called exosomes into their environment. The purpose of this study was to detect MSP proteins in exosomal vesicles of Leishmania spp. protozoa.MethodsUsing mass spectrometry data we determined the profile of MSP class proteins released in L. infantum exosomes derived from promastigotes in their avirulent procyclic (logarithmic) stage and virulent stationary and metacyclic stages. MSP protein isoforms belonging to each of the three msp gene classes could be identified by unique peptides.ResultsMetacyclic promastigote exosomes contained the highest, and logarithmic exosomes had the lowest abundance of total MSP. Among the MSP classes, MSPC class had the greatest variety of isoforms, but was least abundant in all exosomes. Nonetheless, all MSP classes were present at higher levels in exosomes released from stationary or metacyclic promastigotes than logarithmic promastigotes.ConclusionsThe data suggest the efficiency of exosome release may be more important than the identity of MSP isoform in determining the MSP content of Leishmania spp. exosomes.

Failure to detect Tritrichomonas foetus in a cross-sectional survey in the populations of feral cats and owned outpatient cats on St Kitts, West Indies

Objectives For over two decades, feline trichomonosis caused by Tritrichomonas foetus has been recognized as a large-bowel protozoan disease of the domestic cat. It has a wide distribution, but no reports exist in the Caribbean. The objectives of this study were to detect the presence of T foetus and its prevalence in the domestic cat on St Kitts, West Indies. Methods A cross-sectional study was performed between September 2014 and December 2015. This study recruited 115 feral cats from a trap–neuter–return program and 37 owned cats treated as outpatients at the university veterinary clinic. Fresh feces were inoculated in InPouch culture medium, as per the manufacturer’s instructions. In addition, PCR was performed using primers for T foetus. DNA extraction with amplification using primers of a Felis catus NADH dehydrogenase subunit 6 was used as a housekeeping gene for quality control. Results Only two owned cats had reported diarrhea in the preceding 6 months. None of the 152 samples were positive on InPouch culture microscopic examination. Only 35/69 feral cat fecal DNA samples were positive for the housekeeping gene, of which none tested PCR positive for T foetus. Conclusions and relevance T foetus was not detected by culture and PCR in feral cats and owned cats on St Kitts. A high proportion of PCR inhibitors in the DNA samples using a commercial fecal DNA kit can lead to underestimating the prevalence, which should be taken into consideration when a survey on gastrointestinal pathogens depends exclusively on molecular detection.