Rapid Species Diagnosis for Invasive Candidiasis Using Mass Spectrometry

BACKGROUND: Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI TOF-MS) allows the identification of most bacteria and an increasing number of fungi. The potential for the highest clinical benefit of such methods would be in severe acute infections that require prompt treatment adapted to the infecting species. Our objective was to determine whether yeasts could be identified directly from a positive blood culture, avoiding the 1-3 days subculture step currently required before any therapeutic adjustments can be made. METHODOLOGY/PRINCIPAL FINDINGS: Using human blood spiked with Candida albicans to simulate blood cultures, we optimized protocols to obtain MALDI TOF-MS fingerprints where signals from blood proteins are reduced. Simulated cultures elaborated using a set of 12 strains belonging to 6 different species were then tested. Quantifiable spectral differences in the 5000-7400 Da mass range allowed to discriminate between these species and to build a reference database. The validation of the method and the statistical approach to spectral analysis were conducted using individual simulated blood cultures of 36 additional strains (six for each species). Correct identification of the species of these strains was obtained. CONCLUSIONS/SIGNIFICANCE: Direct MALDI TOF-MS analysis of aliquots from positive blood cultures allowed rapid and accurate identification of the main Candida species, thus obviating the need for sub-culturing on specific media. Subsequent to this proof-of-principle demonstration, the method can be extended to other clinically relevant yeast species, and applied to an adequate number of clinical samples in order to establish its potential to improve antimicrobial management of patients with fungemia

Temperature Shift and Host Cell Contact Up-Regulate Sporozoite Expression of Plasmodium falciparum Genes Involved in Hepatocyte Infection

Plasmodium sporozoites are deposited in the skin by Anopheles mosquitoes. They then find their way to the liver, where they specifically invade hepatocytes in which they develop to yield merozoites infective to red blood cells. Relatively little is known of the molecular interactions during these initial obligatory phases of the infection. Recent data suggested that many of the inoculated sporozoites invade hepatocytes an hour or more after the infective bite. We hypothesised that this pre-invasive period in the mammalian host prepares sporozoites for successful hepatocyte infection. Therefore, the genes whose expression becomes modified prior to hepatocyte invasion would be those likely to code for proteins implicated in the subsequent events of invasion and development. We have used P. falciparum sporozoites and their natural host cells, primary human hepatocytes, in in vitro co-culture system as a model for the pre-invasive period. We first established that under co-culture conditions, sporozoites maintain infectivity for an hour or more, in contrast to a drastic loss in infectivity when hepatocytes were not included. Thus, a differential transcriptome of salivary gland sporozoites versus sporozoites co-cultured with hepatocytes was established using a pan-genomic P. falciparum microarray. The expression of 532 genes was found to have been up-regulated following co-culture. A fifth of these genes had no orthologues in the genomes of Plasmodium species used in rodent models of malaria. Quantitative RT-PCR analysis of a selection of 21 genes confirmed the reliability of the microarray data. Time-course analysis further indicated two patterns of up-regulation following sporozoite co-culture, one transient and the other sustained, suggesting roles in hepatocyte invasion and liver stage development, respectively. This was supported by functional studies of four hitherto uncharacterized proteins of which two were shown to be sporozoite surface proteins involved in hepatocyte invasion, while the other two were predominantly expressed during hepatic parasite development. The genome-wide up-regulation of expression observed supports the hypothesis that the shift from the mosquito to the mammalian host contributes to activate quiescent salivary gland sporozoites into a state of readiness for the hepatic stages. Functional studies on four of the up-regulated genes validated our approach as one means to determine the repertoire of proteins implicated during the early events of the Plasmodium infection, and in this case that of P. falciparum, the species responsible for the severest forms of malaria

Couplage orthogonal entre un piège ionique et un analyseur temps de vol IT/O-reTOF

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Preparing for Transmission: Gene Regulation in Plasmodium Sporozoites

International audiencePlasmodium sporozoites are transmitted to mammals by anopheline mosquitoes and first infect the liver, where they transform into replicative exoerythrocytic forms, which subsequently release thousands of merozoites that invade erythrocytes and initiate the malaria disease. In some species, sporozoites can transform into dormant hypnozoites in the liver, which cause malaria relapses upon reactivation. Transmission from the insect vector to a mammalian host is a critical step of the parasite life cycle, and requires tightly regulated gene expression. Sporozoites are formed inside oocysts in the mosquito midgut and become fully infectious after colonization of the insect salivary glands, where they remain quiescent until transmission. Parasite maturation into infectious sporozoites is associated with reprogramming of the sporozoite transcriptome and proteome, which depends on multiple layers of transcriptional and post-transcriptional regulatory mechanisms. An emerging scheme is that gene expression in Plasmodium sporozoites is controlled by alternating waves of transcription activity and translational repression, which shape the parasite RNA and protein repertoires for successful transition from the mosquito vector to the mammalian host

Guidelines for Mathematical Modeling Training of Future Engineers During Classroom Studies on the Theory of Stochastic Processes

Обґрунтовано важливість формування в майбутніх інженерів уміння математичного моделювання під час навчання теорії ймовірностей та випадкових процесів. Розглянуто поняття математичного моделювання та основних його етапів під час навчання студентів технічних ВНЗ математичним дисциплінам. Проаналізовано труднощі, що виникають у студентів під час побудови моделей до професійно орієнтованих та практичних завдань. Запропоновано використання прийомів евристичної діяльності на першому етапі моделювання професійно орієнтованих завдань. Наведено приклад побудови моделі під час лекції до професійно орієнтованого завдання, що ілюструє марковський випадковий процес.The importance of formation of the mathematical modeling ability during the study of the theory of probability and stochastic processes by future engineers is substantiated. The notion of mathematical modeling when teaching the students of technical universities to the mathematical disciplines is examined. The paper reveals the difficulties met by students during the construction of models to the problems. The author notes that the universal formalization algorithm of real problems does not exist; therefore the most difficult for students are the first and the second stages of simulation when solving professionally oriented tasks. In order to solve a problem the techniques of heuristic activity are proposed to take advantage in the first stage of modeling. The study displays one of the ways of teaching students to the «art of modeling», namely the implementation of its development as the ability of students to «see different in the same and the same in differences». This article contains an example of building a model to a professionally oriented task during a lecture. It is shown how a teacher in the course of constructing a model can engage students into interactive debate. For this purpose the teacher’s notation on the blackboard should be accompanied by an appropriate dialogue with students. Methodological recommendations for the direction of educational and cognitive activities of students during the construction of models reflecting Markov random process of discrete state and continuous time are suggested. It is shown why such learning activities, which are to build and study models of stochastic process, contribute to the conscious assimilation of the topic by the students, as well as to the building of their understanding of the unity of some sections of higher mathematics, stochastic processes and connection with real engineering studies. The author emphasizes that the work with a mathematical model of a real engineering process enhances motivation to learn the discipline, so that the students actively master the skills necessary for their future careers. The issue of mathematical modeling training and research of other stochastic processes may become the subject of further survey in this area

Proteomic analysis of Plasmodium sporozoites limited samples using the timsTOF Pro

International audienc

Plasmodium sporozoites on the move: Switching from cell traversal to productive invasion of hepatocytes

International audienceParasites of the genus Plasmodium, the etiological agent of malaria, are transmitted through the bite of anopheline mosquitoes, which deposit sporozoites into the host skin. Sporozoites migrate through the dermis, enter the bloodstream, and rapidly traffic to the liver. They cross the liver sinusoidal barrier and traverse several hepatocytes before switching to productive invasion of a final one for replication inside a parasitophorous vacuole. Cell traversal and productive invasion are functionally independent processes that require proteins secreted from specialized secretory organelles known as micronemes. In this review, we summarize the current understanding of how sporozoites traverse through cells and productively invade hepatocytes, and discuss the role of environmental sensing in switching from a migratory to an invasive state. We propose that timely controlled secretion of distinct microneme subsets could play a key role in successful migration and infection of hepatocytes. A better understanding of these essential biological features of the Plasmodium sporozoite may contribute to the development of new strategies to fight against the very first and asymptomatic stage of malaria

In‐depth proteomic analysis of Plasmodium berghei sporozoites using trapped ion mobility spectrometry with parallel accumulation‐serial fragmentation

International audienceSporozoites of the malaria parasite Plasmodium are transmitted by mosquitoes and infect the liver for an initial and obligatory round of replication, before exponential multiplication in the blood and onset of the disease. Sporozoites and liver stages provide attractive targets for malaria vaccines and prophylactic drugs. In this context, defining the parasite proteome is important to explore the parasite biology and to identify potential targets for antimalarial strategies. Previous studies have determined the total proteome of sporozoites from the two main human malaria parasites, P. falciparum and P. vivax, as well as P. yoelii, which infects rodents. Another murine malaria parasite, P. berghei, is widely used to investigate the parasite biology. However, a deep view of the proteome of P. berghei sporozoites is still missing. To fill this gap, we took advantage of the highly sensitive timsTOF PRO mass spectrometer, combined with three alternative methods for sporozoite purification, to identify the proteome of P. berghei sporozoites using low numbers of parasites. This study provides a reference proteome for P. berghei sporozoites, identifying a core set of proteins expressed across species, and illustrates how the unprecedented sensitivity of the timsTOF PRO system enables deep proteomic analysis from limited sample amounts

Correlation between EphA2 expression level and infection rates.

<p><b>A.</b> Hepa 1–6 cells infected with PbGFP sporozoites were stained 24 hours post-infection with an anti-EphA2 primary antibody (D4A2) followed by Alexa Fluor 594 secondary antibodies (blue histogram), and analyzed by FACS. The grey histogram corresponds to control cells labeled with secondary antibodies only. The vertical line indicates the threshold used to distinguish EphA2^low and EphA2^high cells. <b>B.</b> The percentage of EphA2^low and EphA2^high cells, was determined in non-infected (GFP-negative) and infected (GFP-positive) cells. <b>C.</b> Hepa 1–6 cells infected with PbGFP sporozoites were stained 24 hours post-infection with an anti-CD81 primary antibody (MT81) followed by Alexa Fluor 594 secondary antibodies (blue histogram), and analyzed by FACS. The grey histogram corresponds to control cells labeled with secondary antibodies only. The vertical line indicates the threshold used to distinguish CD81^low and CD81^high cells. This threshold was arbitrarily defined to obtain a majority of CD81^low population after siRNA silencing of CD81. <b>D.</b> The percentage of CD81^low and CD81^high cells, was determined in non-infected (GFP-negative) and infected (GFP-positive) cells. <b>E.</b> Hepa1-6 cells were transfected with siRNA targeting CD81 and infected with PbGFP sporozoites. Cells were stained 24 hours post-infection with an anti-CD81 primary antibody (MT81) followed by Alexa Fluor 594 secondary antibodies (orange histogram), and analyzed by FACS. The grey histogram corresponds to control cells labeled with secondary antibodies only, and the blue histogram corresponds to the control cells labeled with CD81 antibodies, as in C. The vertical line indicates the same threshold as in C, used to distinguish CD81^low and CD81^high cells. <b>F.</b> The percentage of CD81^low and CD81^high cells was determined in non-infected (GFP-negative) and infected (GFP-positive) CD81 siRNA-treated Hepa1-6 cells.</p

EphA2 is not required for sporozoite cell traversal.

<p><b>A-B</b>. Hepa1-6 cells were transfected with siRNA oligonucleotides targeting mouse EphA2 (siEphA2) or with a control siRNA (siCtrl). 48 hours after siRNA transfection, cells were incubated for 1.5 hours with PyGFP (<b>A</b>) or PbGFP (<b>B</b>) sporozoites in the presence of rhodamine-labeled dextran, and the number of traversed (dextran-positive) cells was determined by FACS. NS, non-significant (Mann-Whitney U Test, two-tailed).</p