Something Old, Something New: Ion Channel Blockers as Potential Anti-Tuberculosis Agents.

Tuberculosis (TB) remains a challenging global health concern and claims more than a million lives every year. We lack an effective vaccine and understanding of what constitutes protective immunity against TB to inform rational vaccine design. Moreover, treatment of TB requires prolonged use of multi-drug regimens and is complicated by problems of compliance and drug resistance. While most Mycobacterium tuberculosis (Mtb) bacilli are quickly killed by the drugs, the prolonged course of treatment is required to clear persistent drug-tolerant subpopulations. Mtb's differential sensitivity to drugs is, at least in part, determined by the interaction between the bacilli and different host macrophage populations. Therefore, to design better treatment regimens for TB, we need to understand and modulate the heterogeneity and divergent responses that Mtb bacilli exhibit within macrophages. However, developing drugs de-novo is a long and expensive process. An alternative approach to expedite the development of new TB treatments is to repurpose existing drugs that were developed for other therapeutic purposes if they also possess anti-tuberculosis activity. There is growing interest in the use of immune modulators to supplement current anti-TB drugs by enhancing the host's antimycobacterial responses. Ion channel blocking agents are among the most promising of the host-directed therapeutics. Some ion channel blockers also interfere with the activity of mycobacterial efflux pumps. In this review, we discuss some of the ion channel blockers that have shown promise as potential anti-TB agents.

TZM-gfp cells: a tractable fluorescent tool for analysis of rare and early HIV-1 infection.

Here we describe TZM-gfp, a novel HIV-1 reporter cell derived from the same parental clone JC.53, used previously to generate the widely-utilized indicator cell line TZM-bl. We re-engineered JC.53 cells to express GFP under regulation of HIV Tat and Rev. We characterize the new reporter cell line to show that TZM-gfp cells are equally susceptible to HIV infection, exhibit minimal background signal, and can report HIV infection in rare cells from a bulk population of experimentally-infected human monocyte-derived macrophages. We demonstrate the utility and sensitivity of the cells in detection of even a single HIV-positive macrophage by fluorescence-assisted correlative electron microscopy, using the GFP signal to guide imaging of HIV virions in primary co-culture. Finally, we used TZM-gfp cells for viral capture during co-culture with human peripheral blood mononuclear cells, showing that TZM-gfp can support outgrowth and analyses of patient-derived primary HIV-1 isolates.

Regulation of kinesin-1 activity by the Salmonella enterica effectors PipB2 and SifA.

Salmonella enterica is an intracellular bacterial pathogen. The formation of its replication niche, which is composed of a vacuole associated with a network of membrane tubules, depends on the secretion of a set of bacterial effector proteins whose activities deeply modify the functions of the eukaryotic host cell. By recruiting and regulating the activity of the kinesin-1 molecular motor, Salmonella effectors PipB2 and SifA play an essential role in the formation of the bacterial compartments. In particular, they allow the formation of tubules from the vacuole and their extension along the microtubule cytoskeleton, and thus promote membrane exchanges and nutrient supply. We have developed in vitro and in cellulo assays to better understand the specific role played by these two effectors in the recruitment and regulation of kinesin-1. Our results reveal a specific interaction between the two effectors and indicate that, contrary to what studies on infected cells suggested, interaction with PipB2 is sufficient to relieve the autoinhibition of kinesin-1. Finally, they suggest the involvement of other Salmonella effectors in the control of the activity of this molecular motor.This article has an associated First Person interview with the first author of the paper.

HIV-associated disruption of lung cytokine networks is incompletely restored in asymptomatic HIV-infected Malawian adults on antiretroviral therapy.

Disruption of lung cytokine networks during chronic HIV infection is incompletely restored in individuals on antiretroviral therapy.

Loss of Humoral and Cellular Immunity to Invasive Nontyphoidal Salmonella during Current or Convalescent Plasmodium falciparum Infection in Malawian Children.

Invasive nontyphoidal Salmonella (iNTS) infections are commonly associated with Plasmodium falciparum infections, but the immunologic basis for this linkage is poorly understood. We hypothesized that P. falciparum infection compromises the humoral and cellular immunity of the host to NTS, which increases the susceptibility of the host to iNTS infection. We prospectively recruited children aged between 6 and 60 months at a Community Health Centre in Blantyre, Malawi, and allocated them to the following groups; febrile with uncomplicated malaria, febrile malaria negative, and nonfebrile malaria negative. Levels of Salmonella enterica serovar Typhimurium-specific serum bactericidal activity (SBA) and whole-blood bactericidal activity (WBBA), complement C3 deposition, and neutrophil respiratory burst activity (NRBA) were measured. Levels of SBA with respect to S Typhimurium were reduced in febrile P. falciparum-infected children (median, -0.20 log10 [interquartile range {IQR}, -1.85, 0.32]) compared to nonfebrile malaria-negative children (median, -1.42 log10 [IQR, -2.0, -0.47], P = 0.052). In relation to SBA, C3 deposition on S Typhimurium was significantly reduced in febrile P. falciparum-infected children (median, 7.5% [IQR, 4.1, 15.0]) compared to nonfebrile malaria-negative children (median, 29% [IQR, 11.8, 48.0], P = 0.048). WBBA with respect to S Typhimurium was significantly reduced in febrile P. falciparum-infected children (median, 0.25 log10 [IQR, -0.73, 1.13], P = 0.0001) compared to nonfebrile malaria-negative children (median, -1.0 log10 [IQR, -1.68, -0.16]). In relation to WBBA, S Typhimurium-specific NRBA was reduced in febrile P. falciparum-infected children (median, 8.8% [IQR, 3.7, 20], P = 0.0001) compared to nonfebrile malaria-negative children (median, 40.5% [IQR, 33, 65.8]). P. falciparum infection impairs humoral and cellular immunity to S Typhimurium in children during malaria episodes, which may explain the increased risk of iNTS observed in children from settings of malaria endemicity. The mechanisms underlying humoral immunity impairment are incompletely understood and should be explored further.

Differential PfEMP1 expression is associated with cerebral malaria pathology.

Plasmodium falciparum is unique among human malarias in its ability to sequester in post-capillary venules of host organs. The main variant antigens implicated are the P. falciparum erythrocyte membrane protein 1 (PfEMP1), which can be divided into three major groups (A-C). Our study was a unique examination of sequestered populations of parasites for genetic background and expression of PfEMP1 groups. We collected post-mortem tissue from twenty paediatric hosts with pathologically different forms of cerebral malaria (CM1 and CM2) and parasitaemic controls (PC) to directly examine sequestered populations of parasites in the brain, heart and gut. Use of two different techniques to investigate this question produced divergent results. By quantitative PCR, group A var genes were upregulated in all three organs of CM2 and PC cases. In contrast, in CM1 infections displaying high levels of sequestration but negligible vascular pathology, there was high expression of group B var. Cloning and sequencing of var transcript tags from the same samples indicated a uniformly low expression of group A-like var. Generally, within an organ sample, 1-2 sequences were expressed at dominant levels. 23% of var tags were detected in multiple patients despite the P. falciparum infections being genetically distinct, and two tags were observed in up to seven hosts each with high expression in the brains of 3-4 patients. This study is a novel examination of the sequestered parasites responsible for fatal cerebral malaria and describes expression patterns of the major cytoadherence ligand in three organ-derived populations and three pathological states.

A simple protocol for platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes in a resource poor setting.

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules (1). By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes (2) and restrict their environment to a more favorable low oxygen pressure (3). As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood. Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria (4,5). The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor (6). Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)(7,8) has also been associated with severe disease (9). One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps (10). Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM (11). In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian (12) and Mozambican patients (13), (although not in Malian (14)). With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay (15). Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

Live imaging of mitosomes and hydrogenosomes by HaloTag technology.

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes.