Reversing BCG-mediated autophagy inhibition and mycobacterial survival to improve vaccine efficacy.

BACKGROUND: Autophagy is an important mechanism for promoting Mycobacterium clearance from macrophages. Pathogenic and non-pathogenic mycobacterium can activate the mTOR pathway while simultaneously inducing autophagy. M. tuberculosis and M. bovis BCG inhibit autophagy and favor intracellular bacteria survival. RESULTS: We observed that pre-infection of live or heat-killed BCG could prevent autophagy induced by pharmacological activators or M. smegmatis, a strong autophagy-inducing mycobacterium. BCG-derived lipids are responsible for autophagy inhibition. However, post-infection with BCG could not stop the autophagy initiated by M. smegmatis, which increases further autophagy induction and mycobacteria clearance. Coinfection with BCG and heat killed M. smegmatis enhanced antigen specific CD4+ T cell responses and reduced mycobacterial survival. CONCLUSION: These results suggest that autophagy-inducing M. smegmatis could be used to promote better innate and consequential adaptive immune responses, improving BCG vaccine efficacy.

Host phospholipase C-γ1 impairs phagocytosis and killing of mycobacteria by J774A.1 murine macrophages.

Macrophages represent the first line of defense against invading Mycobacterium tuberculosis (Mtb). In order to enhance intracellular survival, Mtb targets various components of the host signaling pathways to limit macrophage functions. The outcome of Mtb infection depends on various factors derived from both host and pathogen. A detailed understanding of such factors operating during interaction of the pathogen with the host is a prerequisite for designing new approaches for combating mycobacterial infections. This work analyzed the role of host phospholipase C-γ1 (PLC-γ1) in regulating mycobacterial uptake and killing by J774A.1 murine macrophages. Small interfering RNA mediated knockdown of PLC-γ1 increased internalization and reduced the intracellular survival of both Mtb and Mycobacterium smegmatis (MS) by macrophages. Down-regulation of the host PLC-γ1 was observed during the course of mycobacterial infection within these macrophages. Finally, Mtb infection also suppressed the expression of pro-inflammatory cytokine tumor necrosis factor-α and chemokine (C-C motif) ligand 5 (RANTES) which was restored by knocking down PLC-γ1 in J774A.1 cells. These observations suggest a role of host PLC-γ1 in the uptake and killing of mycobacteria by murine macrophages.

Phospholipase C-γ2 promotes intracellular survival of mycobacteria.

Mycobacterium tuberculosis (Mtb) infects millions of people each year. These bacilli can survive inside macrophages. To favor their survival, pathogen alters various signal transduction pathways in host cells. Phospholipase C (PLC) signaling regulates various processes in mammalian cells but has never been investigated for their roles in regulating phagocytosis and killing of mycobacteria by macrophages. Here, we report that infection with Mtb but not Mycobacterium smegmatis (MS) induces phosphorylation of PLC-γ2 at tyrosine 1217 in J774A.1 cells. Small interfering RNA-mediated knockdown of PLC-γ2 expression leads to the enhanced killing of both MS and Mtb by these cells suggesting that Mtb activates PLC-γ2 to promote its intracellular survival within macrophages. Knockdown of PLC-γ2 also lead to increased uptake of Mtb but not MS by J774.A.1 cells. Further, we have observed that PLC-γ2 was required for Mtb-induced inhibition of expression of proinflammatory cytokine tumor necrosis factor-α, inducible nitric oxide synthase, and chemokine (C-C motif) ligand 5 (RANTES). Altogether, our results for the first time demonstrate that Mtb induces activation of macrophages PLC-γ2 to inhibit their mycobactericidal response.

PknG supports mycobacterial adaptation in acidic environment.

Mycobacterium tuberculosis (Mtb), causative agent of human tuberculosis (TB), has the remarkable ability to adapt to the hostile environment inside host cells. Eleven eukaryotic like serine-threonine protein kinases (STPKs) are present in Mtb. Protein kinase G (PknG) has been shown to promote mycobacterial survival inside host cells. A homolog of PknG is also present in Mycobacterium smegmatis (MS), a fast grower, non-pathogenic mycobacterium. In the present study, we have analyzed the role of PknG in mycobacteria during exposure to acidic environment. Expression of pknG in MS was decreased in acidic medium. Recombinant MS ectopically expressing pknG (MS-G) showed higher growth in acidic medium compared to wild type counterpart. MS-G also showed higher resistance upon exposure to 3.0 pH and better adaptability to acidic pH. Western blot analysis showed differential threonine but not serine phosphorylation of cellular proteins in MS at acidic pH which was restored by ectopic expression of pknG in MS. In Mtb H37Ra (Mtb-Ra), expression of pknG was increased at acidic pH. We also observed decreased expression of pknG in MS during infection in macrophages while the expression of pknG in Mtb-Ra was increased in similar conditions. Taken together, our data strongly suggests that pknG regulates growth of mycobacteria in acidic environment and is differentially transcribed in MS and Mtb under these conditions.

PDCD4 as a marker of mTOR pathway activation and therapeutic target in mycobacterial infections.

Programmed cell death protein 4 (PDCD4) is instrumental in regulating a range of cellular processes such as translation, apoptosis, signal transduction, and inflammatory responses. There is a notable inverse correlation between PDCD4 and the mammalian target of rapamycin (mTOR) pathway, which is integral to cellular growth control. Activation of mTOR is associated with the degradation of PDCD4. Although the role of PDCD4 is well established in oncogenesis and immune response regulation, its function in mycobacterial infections and its interplay with the mTOR pathway necessitate further elucidation. This study investigates the modulation of PDCD4 expression in the context of mycobacterial infections, revealing a consistent pattern of downregulation across diverse mycobacterial species. This observation underscores the potential utility of PDCD4 as a biomarker for assessing mTOR pathway activation during such infections. Building on this finding, we employed a novel approach using PDCD4-based mTOR (Tor)-signal-indicator (TOSI) reporter cells for the high-throughput screening of FDA-approved drugs, focusing on mTOR inhibitors. This methodology facilitated the identification of several agents, inclusive of known mTOR inhibitors, which upregulated PDCD4 expression and concurrently exhibited efficacy in impeding mycobacterial proliferation within macrophages. These results not only reinforce the significance of PDCD4 as a pivotal marker in the understanding of infectious diseases, particularly mycobacterial infections, but also illuminate its potential in the identification of mTOR inhibitors, thereby contributing to the advancement of therapeutic strategies. IMPORTANCE: This study emphasizes the critical role of the mammalian target of rapamycin (mTOR) pathway in macrophage responses to mycobacterial infections, elucidating how mycobacteria activate mTOR, resulting in PDCD4 degradation. The utilization of the (Tor)-signal-indicator (TOSI) vector for real-time monitoring of mTOR activity represents a significant advancement in understanding mTOR regulation during mycobacterial infection. These findings deepen our comprehension of mycobacteria's innate immune mechanisms and introduce PDCD4 as a novel marker for mTOR activity in infectious diseases. Importantly, this research laid the groundwork for high-throughput screening of mTOR inhibitors using FDA-approved drugs, offering the potential for repurposing treatments against mycobacterial infections. The identification of drugs that inhibit mTOR activation opens new avenues for host-directed therapies, marking a significant step forward in combating tuberculosis and other mycobacterial diseases.

Unraveling factors responsible for pathogenic differences in Lassa virus strains.

Lassa virus (LASV) is the etiological agent of Lassa fever (LF), a severe hemorrhagic disease with potential for lethal outcomes. Apart from acute symptoms, LF survivors often endure long-term complications, notably hearing loss, which significantly impacts their quality of life and socioeconomic status in endemic regions of West Africa. Classified as a Risk Group 4 agent, LASV poses a substantial public health threat in affected areas. Our laboratory previously developed a novel lethal guinea pig model of LF utilizing the clinical isolate LASV strain LF2384. However, the specific pathogenic factors underlying LF2384 infection in guinea pigs remained elusive. In this study, we aimed to elucidate the differences in the immunological response induced by LF2384 and LF2350, another LASV isolate from a non-lethal LF case within the same outbreak. Through comprehensive immunological gene profiling, we compared the expression kinetics of key genes in guinea pigs infected with LASV LF2384 and LF2350. Our analysis revealed differential expression patterns for several immunological genes, including CD94, CD19-2, CD23, IL-7, and CIITA, during LF2384 and LF2350 infection. Moreover, through the generation of recombinant LASVs, we sought to identify the specific viral genes responsible for the observed pathogenic differences between LF2384 and LF2350. Our investigations pinpointed the L protein as a crucial determinant of pathogenicity in guinea pigs infected with LASV LF2384.

Comparison of Analytical Sensitivity (Limit of Detection) of Xpert MTB/RIF and Xpert MTB/RIF Ultra for Non-Sputum Specimens.

Tuberculosis (TB) is a significant public health threat and has remained a leading cause of death in many parts of the world. Rapid and accurate testing and timely diagnosis can improve treatment efficacy and reduce new exposures. The Cepheid Xpert® MTB/RIF tests have two marketed products (US-IVD and Ultra) that are widely accepted for diagnosis of TB but have not yet been approved for non-sputum specimens. Despite numerous studies in the literature, no data for the analytical sensitivity of these two products on the non-sputum samples are available to date. This is the first study that systematically determined the analytical sensitivities of both US-IVD and Ultra tests on cerebrospinal fluid (CSF), tissue, and bronchoalveolar lavage (BAL). The limits of detection (LoDs) on the US-IVD test for both Mycobacterium tuberculosis and rifampin resistance in CFU/mL, respectively, were as follows: CSF (3.3 and 4.6), tissue (15 and 23), and bronchoalveolar lavage (BAL) (45 and 60), and on the Ultra test: CSF (0.16 and 2.7), tissue (0.11 and 12), and BAL (0.65, and 7.5). Overall, the analytical sensitivities of the Ultra test were substantially better than US-IVD for all sample types tested. This study provided a foundation for using either the US-IVD or Ultra test for the early detection of both pulmonary and extrapulmonary (EP) TB. Furthermore, using Ultra could result in higher TB case detection rates in subjects with paucibacillary TB and EP TB, positively impacting WHO goals to eradicate TB.

Synthesis and Rational design of Europium and Lithium Doped Sodium Zinc Molybdate with Red Emission for Optical Imaging.

Highly efficient fluorescent and biocompatible europium doped sodium zinc molybdate (NZMOE) nanoprobes were successfully synthesized via Polyol method. Non-radiative defect centres get reduced with Li+ co-doping in NZMOE nanoprobes. XRD spectra and Rietveld refinement confirmed successful incorporation of lithium ion and crystallinity was also improved with Li+ co-doping. The shape of phosphor is rod shaped, as determined by TEM. Significant enhancement in photoluminescence intensity was observed with 266, 395 and 465 nm excitations. Profound red emission was recorded for 5 at% Li+ co-doped NZMOE nanoprobes with 266 nm excitation. It shows high asymmetry ratio (~15), color purity (94.90%) and good quantum efficiency (~70%). Judd Ofelt parameters have been calculated to measure intensity parameters and radiative transition rates. In order to measure biocompatibility of the nanoprobes, cytotoxicity assays were performed with HePG2 cells. The fluorescence emitted from phosphor material treated HePG2 cells was also measured by Laser Scanning Confocal Microscopy. The bright red fluorescence in HePG2 cells treated with very low concentration (20 µg/ml) of phosphor material indicates that it could be a promising phosphor for biological detection or bio-imaging.