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.

Tuberculosis: Smart manipulation of a lethal host.

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a global threat to human health. Development of drug resistance and co-infection with HIV has increased the morbidity and mortality caused by TB. Macrophages serve as primary defense against microbial infections, including TB. Upon recognition and uptake of mycobacteria, macrophages initiate a series of events designed to lead to generation of effective immune responses and clearance of infection. However, pathogenic mycobacteria utilize multiple mechanisms for manipulating macrophage responses to protect itself from being killed and to survive within these cells that are designed to kill them. The outcomes of mycobacterial infection are determined by several host- and pathogen-related factors. Significant advancements in understanding mycobacterial pathogenesis have been made in recent years. In this review, some of the important factors/mechanisms regulating mycobacterial survival inside macrophages are discussed.

Phospholipase C: underrated players in microbial infections.

During bacterial infections, one or more virulence factors are required to support the survival, growth, and colonization of the pathogen within the host, leading to the symptomatic characteristic of the disease. The outcome of bacterial infections is determined by several factors from both host as well as pathogen origin. Proteins and enzymes involved in cellular signaling are important players in determining the outcome of host-pathogen interactions. phospholipase C (PLCs) participate in cellular signaling and regulation by virtue of their ability to hydrolyze membrane phospholipids into di-acyl-glycerol (DAG) and inositol triphosphate (IP3), which further causes the activation of other signaling pathways involved in various processes, including immune response. A total of 13 PLC isoforms are known so far, differing in their structure, regulation, and tissue-specific distribution. Different PLC isoforms have been implicated in various diseases, including cancer and infectious diseases; however, their roles in infectious diseases are not clearly understood. Many studies have suggested the prominent roles of both host and pathogen-derived PLCs during infections. PLCs have also been shown to contribute towards disease pathogenesis and the onset of disease symptoms. In this review, we have discussed the contribution of PLCs as a determinant of the outcome of host-pathogen interaction and pathogenesis during bacterial infections of human importance.

Role of Streptococcus pneumoniae extracellular glycosidases in immune evasion.

Streptococcus pneumoniae (pneumococcus) typically colonizes the human upper airway asymptomatically but upon reaching other sites of the host body can cause an array of diseases such as pneumonia, bacteremia, otitis media, and meningitis. Be it colonization or progression to disease state, pneumococcus faces multiple challenges posed by host immunity ranging from complement mediated killing to inflammation driven recruitment of bactericidal cells for the containment of the pathogen. Pneumococcus has evolved several mechanisms to evade the host inflicted immune attack. The major pneumococcal virulence factor, the polysaccharide capsule helps protect the bacteria from complement mediated opsonophagocytic killing. Another important group of pneumococcal proteins which help bacteria to establish and thrive in the host environment is surface associated glycosidases. These enzymes can hydrolyze host glycans on glycoproteins, glycolipids, and glycosaminoglycans and consequently help bacteria acquire carbohydrates for growth. Many of these glycosidases directly or indirectly facilitate bacterial adherence and are known to modulate the function of host defense/immune proteins likely by removing glycans and thereby affecting their stability and/or function. Furthermore, these enzymes are known to contribute the formation of biofilms, the bacterial communities inherently resilient to antimicrobials and host immune attack. In this review, we summarize the role of these enzymes in host immune evasion.

Functional characterization delineates that a Mycobacterium tuberculosis specific protein kinase (Rv3080c) is responsible for the growth, phagocytosis and intracellular survival of avirulent mycobacteria.

Serine/threonine protein kinases (STPKs) are predominantly involved in growth, development, division, differentiation, and in regulating immune responses in mycobacteria. A wide variety of functions of mycobacterial STPKs persuade mycobacterial growth and further its survival in the hosts. The polymorphic studies have shown that a full length gene of Rv3080c (pknK) is present in the slow growing mycobacteria. The wild type Mycobacterium smegmatis containing only vector (M. smegmatis) and M. smegmatis containing Rv3080c (pknK) cloned in pMV261 vector (M. smegmatis::K) were cultured in different growth media. The studies have shown that M. smegmatis did not differ in the growth and in survival while a substantial reduction in the growth (four-ten-folds) and a significant delay in the colony formation were observed in M. smegmatis::K. In order to look for the stage specific and modulated expression of PknK, the study was comprehended to quantitate pknK transcripts at different phases of cultures. The mycobacterium, containing high copy number of pknK specific RNA was unable to multiply. The study thus highlights that Rv3080c is largely accountable for changing the fate of avirulent mycobacteria and hence the protein can be utilized as an important molecule to target pathogenesis.

Mycobacterial response to an acidic environment: protective mechanisms.

Given the emergence and spread of multidrug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb), the world faces the urgency of finding new drugs to combat tuberculosis. Understanding the biochemical/physiological processes enabling Mtb to survive the stressful environment within macrophages and acquire tolerance, resistance and persistence against the stresses are the key to developing new approaches to tackle this health problem. As Mtb gains entry into the respiratory tract and is engulfed by macrophages, lowering pH acts as a primary defence of phagosomes within macrophages and also in the centres of caseating granulomas. It becomes essential for the pathogen to maintain pH homeostasis for survival in these conditions. Acid resistance mechanisms are well known and extensively studied in other bacteria such as Escherichia coli, Lactobacillus spp., Brucella spp., Helicobacter pylori and Listeria monocytogenes. However, in the case of Mtb, acid tolerance and resistance mechanisms still need to be explored in detail. This review aims to describe the current understanding of underlying mechanisms involved in countering low pH faced by Mtb as the acid resistance/tolerance mechanisms contribute to the pathogenesis of the disease.

Laboratory diagnosis of COVID-19: current status and challenges.

The magnitude and pace of global affliction caused by Coronavirus Disease-19 (COVID-19) is unprecedented in the recent past. From starting in a busy seafood market in the Chinese city of Wuhan, the virus has spread across the globe in less than a year, infecting over 76 million people and causing death of close to 1.7 million individuals worldwide. As no specific antiviral treatment is currently available, the major strategy in containing the pandemic is focused on early diagnosis and prompt isolation of the infected individuals. Several diagnostic modalities have emerged within a relatively short period, which can be broadly classified into molecular and immunological assays. While the former category is centered around real-time PCR, which is currently considered the gold standard of diagnosis, the latter aims to detect viral antigens or antibodies specific to the viral antigens and is yet to be recommended as a stand-alone diagnostic tool. This review aims to provide an update on the different diagnostic modalities that are currently being used in diagnostic laboratories across the world as well as the upcoming methods and challenges associated with each of them. In a rapidly evolving diagnostic landscape with several testing platforms going through various phases of development and/or regulatory clearance, it is prudent that the clinical community familiarizes itself with the nuances of different testing modalities currently being employed for this condition.

Downregulation of protein kinase C-alpha enhances intracellular survival of Mycobacteria: role of PknG.

BACKGROUND: Intracellular trafficking of mycobacteria is comprehensively dependent on the unusual regulation of host proteins. Recently, we have reported that infection of macrophages by Mycobacterium tuberculosis H37Rv (Rv) selectively downregulates the expression of PKCalpha while infection by Mycobacterium smegmatis (MS) does not. RESULTS: Based on our earlier study, we have extrapolated for the first time that knockdown of PKCalpha, impairs phagocytosis of mycobacteria by macrophages while their intracellular survival is drastically increased. Mycobacterium bovis BCG (BCG) and Mycobacterium tuberculosis H37Ra (Ra) have also been shown to downregulate the expression of PKCalpha during the infection. Since PknG is uniquely expressed in BCG, Ra, Rv but not in MS and has been reported to promote intracellular survival of mycobacteria, led us to believe that PknG may be involved in such downregulation of PKCalpha. THP-1 cells infected with recombinant MS expressing PknG (MS-G), showed significant reduction in PKCalpha expression. In normal THP-1 cells survival of MS-G was enhanced as compared to MS, while their behavior in PKCalpha deficient cells could not be distinguished. The results strongly demonstrate that pathogenic mycobacteria recognize and then inhibit PKCalpha to circumvent phagocytosis and the hostile environment of macrophages. We emphasize that, this inhibition is controlled by PknG. CONCLUSIONS: All together, our data reveal a mechanism that shows substantial interdependence of PKCalpha with PknG, in sustaining mycobacterial infection.

Intrinsically Disordered Human T Lymphotropic Virus Type 1 p30 Protein: Experimental and Computational Evidence.

Human T lymphotropic virus type 1 (HTLV-1) causes adult T cell leukemia and lymphoma and other neuroinflammatory diseases. The pX region of HTLV-1 genome encodes an accessory protein p30 that is required for viral persistence and spread in the host. p30 regulates viral gene expression at the transcription level by competing with Tax for p300 binding and at posttranscriptional level by nuclear retention of tax/rex messenger RNA (mRNA). In addition, p30 modulates the host cellular environment by binding to various host proteins such as ATM, REGγ, and PRMT5. However, the low expression levels of p30 has been a major hurdle in studying its structure-function relationship in the context of HTLV-1 pathobiology, which is most likely due to its intrinsically disordered nature. To investigate the unstable nature of p30, flow cytometric analysis of p30-GFP fusion protein expressed in Escherichia coli was conducted and bioinformatics analysis of p30 was performed. The bacterial cells were green fluorescent protein (GFP) positive, indicating that p30-GFP was in the soluble fraction. Induction, particularly at higher temperature, reduced the expression of p30-GFP. Moreover, p30-GFP was detected exclusively in insoluble fraction upon cell lysis, suggesting its unstable and disordered nature. The bioinformatics analysis of p30 protein sequence and amino acid content revealed that p30 has highly disordered regions from amino acids 75-155 and 197-241. Furthermore, p30 has regions for macromolecular interactions that could stabilize it and these regions coincide with the unstable regions. Collectively, the study indicates that HTLV-1 p30 is an intrinsically disordered protein.

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.

Differential regulation of protein kinase C isoforms of macrophages by pathogenic and non-pathogenic mycobacteria.

Given the fact that Mycobacterium tuberculosis (Mtb) may respond to the intracellular milieu of the macrophage with the induction of environmentally regulated genes required for survival and growth of the bacteria we assumed that the protein kinases may also be the factors in Mycobacterium-macrophage interaction. Since, protein kinases play a major role in various critical cellular processes including regulation of immune responses, we describe the fate of expression and phosphorylation of protein kinase C in macrophage cell lines exposed to Mtb H37Rv and raised the question whether the change in the events of expression and phosphorylation are the results of direct interaction of bacilli with macrophages and/or, are also indirectly mediated by specific cytokines that are induced in response to exposure. Our results show that only novel PKCs are phosphorylated during infection of macrophages by pathogenic and non-pathogenic mycobacteria and the alteration is a result of direct host-bacilli association which is independent of cytokines as mediators. Expression of PKC-alpha (conventional PKC isoform) was down regulated by Mtb H37Rv. In contrast the non-pathogenic fast grower Mycobacterium smegmatis (MS) increased the expression and phosphorylation of PKC-alpha. PKC-alpha was also increased in macrophages treated with serum of mice immunized with Mtb H37Rv. The study has shown that pathogenic and non-pathogenic mycobacteria categorically select the type of protein kinases C for activation/deactivation.