In vitro screening of natural product-based compounds for leishmanicidal activity.

Leishmaniasis is one of the major parasitic diseases, caused by obligate intracellular protozoa Leishmania, having high mortality as well as morbidity rate. As there is no human licensed vaccine available against leishmaniasis, chemotherapy remains the major way of combating this disease. Many disadvantages are known to be associated with the current drug regime including severe side effects and toxicity, long duration and expensive treatment, and the emergence of resistance. An alternative approach is being utilized to search for active molecules using natural sources, rather than relying on synthetic drugs. Many plant-derived secondary metabolites like phenolic compounds, steroids, quinones, etc. are being extensively investigated for their anti-leishmanial potential. One such group of complex phenolic compounds are diarylheptanoids. These compounds have been shown to exhibit anti-inflammatory, anti-parasitic, anti-fungal, and other pharmacological activities. In the present study, a set of sixteen tetrahydropyran derivatives including three natural products were obtained in lyophilized form. These compounds with trans-2,6-disubstituted tetrahydropyrans, Diospongin A, Diospongin B (isolated from Dioscorea spongiosa) and Centrolobine (Centrolobium sclerophyllum) as parent compounds were synthesized by the reaction of 1-phenyl-1-triemthylsiloxyethylene with six-membered cyclic hemiacetals in the presence of iodine as a catalyst. All the sixteen synthesized tetrahydropyran derivatives were used for toxicity analysis against L. donovani promastigotes, amastigotes and THP-1-derived human macrophages. IC50 values and selectivity index were calculated for all the compounds. Out of these sixteen, five compounds showed the best effect in vitro in terms of both leishmanicidal activity and non-toxicity to human macrophages.

Rapid diagnosis of Plasmodium falciparum malaria using a point-of-care loop-mediated isothermal amplification device.

LAMP diagnosis of malaria is simple and cost-effective with acceptable sensitivity and specificity as compared to standard diagnostic modules such as microscopy, RDTs and nested PCR, and thus its deployment for onsite screening of malaria in resource-limited regions is under consideration. However, the requirement of an electricity-operated dry bath and bulky read-out unit is still a major concern. In an effort to simplify this limitation, we have developed a portable LAMP device and fluorescence readout unit which can be used in the rapid point-of-care diagnosis of malaria. We have developed a point-of-care diagnostic LAMP device that is easy to operate by a mobile application, and the results can be quantified with a fluorescent readout unit. The diagnostic performance of the device was evaluated in 90 P. falciparum-infected clinical isolates stored at 4°C for 6-7 years and 10 freshly collected isolates from healthy volunteers. The LOD and quantitative ability of LAMP in estimating parasitemia levels were revealed with laboratory-grown P. falciparum strain (3D7). The LAMP assay performed in our device was exclusive for P. falciparum detection with sensitivity and specificity determined to be 98.89% and 100%, respectively, in clinical isolates. The LOD was documented to be 1 parasite/µl at the cut-off ADC value of 20. Parasite density estimated from ADC values showed concordance with microscopically determined parasite density of the cultured P. falciparum 3D7 strain. The LAMP assay performed in our device provides a possible portable platform for its deployment in the point-of-care diagnosis of malaria. Further validation of the quantitative ability of the assay with freshly collected or properly stored clinical samples of known parasitemia is necessary for field applicability.

Rapid diagnosis of Leishmania infection with a portable loopmediated isothermal amplification device.

L. donovani is an intracellular protozoan parasite, that causes visceral leishmaniasis (VL), and consequently, post-kala azar dermal leishmaniasis (PKDL). Diagnosis and treatment of leishmaniasis is crucial for decreasing its transmission. Various diagnostic techniques like microscopy, enzyme-linked immunosorbent assays (ELISA) and PCR-based methods are used to detect leishmaniasis infection. More recently, loop-mediated isothermal amplification (LAMP) assay has emerged as an ideal diagnostic measure for leishmaniasis, primarily due to its accuracy, speed and simplicity. However, point-of-care diagnosis is still not been tested with the LAMP assay. We have developed a portable LAMP device for the monitoring of Leishmania infection. The LAMP assay performed using our device can detect and amplify as little as 100 femtograms of L. donovani DNA. In a preliminary study, we have shown that the device can also amplify L. donovani DNA present in VL and PKDL patient samples with high sensitivity (100%), specificity (98%) and accuracy (99%), and can be used both for diagnostic and prognostic analysis. To our knowledge, this is the first report to describe the development and application of a portable LAMP device which has the potential to evolve as a point-of-care diagnostic and prognostic tool for Leishmania infections in future.

Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

The arginine sensing and transport binding sites are distinct in the human pathogen Leishmania.

The intracellular protozoan parasite Leishmania donovani causes human visceral leishmaniasis. Intracellular L. donovani that proliferate inside macrophage phagolysosomes compete with the host for arginine, creating a situation that endangers parasite survival. Parasites have a sensor that upon arginine deficiency activates an Arginine Deprivation Response (ADR). L. donovani transport arginine via a high-affinity transporter (LdAAP3) that is rapidly up-regulated by ADR in intracellular amastigotes. To date, the sensor and its ligand have not been identified. Here, we show that the conserved amidino group at the distal cap of the arginine side chain is the ligand that activates ADR, in both promastigotes and intracellular amastigotes, and that arginine sensing and transport binding sites are distinct in L. donovani. Finally, upon addition of arginine and analogues to deprived cells, the amidino ligand activates rapid degradation of LdAAP3. This study provides the first identification of an intra-molecular ligand of a sensor that acts during infection.

Listeriolysin O Regulates the Expression of Optineurin, an Autophagy Adaptor That Inhibits the Growth of Listeria monocytogenes.

Autophagy, a well-established defense mechanism, enables the elimination of intracellular pathogens including Listeria monocytogenes. Host cell recognition results in ubiquitination of L. monocytogenes and interaction with autophagy adaptors p62/SQSTM1 and NDP52, which target bacteria to autophagosomes by binding to microtubule-associated protein 1 light chain 3 (LC3). Although studies have indicated that L. monocytogenes induces autophagy, the significance of this process in the infectious cycle and the mechanisms involved remain poorly understood. Here, we examined the role of the autophagy adaptor optineurin (OPTN), the phosphorylation of which by the TANK binding kinase 1 (TBK1) enhances its affinity for LC3 and promotes autophagosomal degradation, during L. monocytogenes infection. In LC3- and OPTN-depleted host cells, intracellular replicating L. monocytogenes increased, an effect not seen with a mutant lacking the pore-forming toxin listeriolysin O (LLO). LLO induced the production of OPTN. In host cells expressing an inactive TBK1, bacterial replication was also inhibited. Our studies have uncovered an OPTN-dependent pathway in which L. monocytogenes uses LLO to restrict bacterial growth. Hence, manipulation of autophagy by L. monocytogenes, either through induction or evasion, represents a key event in its intracellular life style and could lead to either cytosolic growth or persistence in intracellular vacuolar structures.

ActA of Listeria monocytogenes and Its Manifold Activities as an Important Listerial Virulence Factor.

Listeria monocytogenes is a ubiquitously occurring gram-positive bacterium in the environment that causes listeriosis, one of the deadliest foodborne infections known today. It is a versatile facultative intracellular pathogen capable of growth within the host's cytosolic compartment. Following entry into the host cell, L. monocytogenes escapes from vacuolar compartments to the cytosol, where the bacterium begins a remarkable journey within the host cytoplasm, culminating in bacterial spread from cell to cell, to deeper tissues and organs. This dissemination process depends on the ability of the bacterium to harness central components of the host cell actin cytoskeleton using the surface bound bacterial factor ActA (actin assembly inducing protein). Hence ActA plays a major role in listerial virulence, and its absence renders bacteria intracellularly immotile and essentially non-infectious. As the bacterium, moving by building a network of filamentous actin behind itself that is often referred to as its actin tail, encounters cell-cell contacts it forms double-vacuolar protrusions that allow it to enter the neighboring cell where the cycle then continues. Recent studies have now implicated ActA in other stages of the life cycle of L. monocytogenes. These include extracellular properties of aggregation and biofilm formation to mediate colonization of the gut lumen, promotion and enhancement of bacterial host cell entry, evasion of autophagy, vacuolar exit, as well as nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) activation. These novel properties provide a new view of ActA and help explain its role as an essential virulence factor of L. monocytogenes.