Seyed E. Hasnain

The PE/PPE multigene family codes for virulence factors and is a possible source of mycobacterial antigenic variation: perhaps more?

The PE/PPE multigene family codes for approximately 10% of the Mycobacterium tuberculosis proteome and is encoded by 176 open reading frames. These proteins possess, and have been named after, the conserved proline-glutamate (PE) or proline-proline-glutamate (PPE) motifs at their N-terminus. Their genes have a conserved structure and repeat motifs that could be a potential source of antigenic variation in M. tuberculosis. PE/PPE genes are scattered throughout the genome and PE/PPE pairs are usually encoded in bicistronic operons although this is not universally so. This gene family has evolved by specific gene duplication events. PE/PPE proteins are either secreted or localized to the cell surface. Several are thought to be virulence factors, which participate in evasion of the host immune response. This review summarizes the current knowledge about the gene family in order to better understand its biological function.

Mycobacterium tuberculosis PE25/PPE41 protein complex induces necrosis in macrophages: Role in virulence and disease reactivation?

Necrotic cell death during TB infection is an important prerequisite for bacterial dissemination and virulence. The underlying mechanisms and the bacterial factors involved therein are not well understood. The Mycobacterium tuberculosis (M. tuberculosis) co‐operonic PE25/PPE41 protein complex, similar to ESAT‐6/CFP‐10, belonging to the PE/PPE and ESAT‐6 families of genes has co‐expanded and co‐evolved in the genomes of pathogenic mycobacteria. We report a novel role of this highly immunogenic PE25/PPE41 protein complex in inducing necrosis, but not apoptosis, in macrophages. We propose that these protein complexes of M. tuberculosis, secreted by similar/unique transport system (Type VII), have an important role in M. tuberculosis virulence and disease reactivation.

Dormancy Associated Translation Inhibitor (DATIN/Rv0079) of Mycobacterium tuberculosis interacts with TLR2 and induces proinflammatory cytokine expression

Mycobacterium tuberculosis, the cause of tuberculosis in humans, is present approximately in one third of the worlds population, mostly in a dormant state. The proteins encoded by the dormancy survival regulon (DosR regulon) are mainly responsible for survival of the bacilli in a latent form. To maintain latency, mycobacteria orchestrate a balanced interplay of different cytokines secreted by immune cells during the granulomatous stage. The function of most of the DosR regulon proteins of M. tuberculosis is unknown. In this study, we have shown that one of the DosR regulon proteins, DATIN, encoded by the gene Rv0079, can stimulate macrophages and peripheral blood mononuclear cells (PBMC) to secrete important cytokines that may be significant in granuloma formation and its maintenance. The expression level of DATIN in Mycobacterium bovis BCG was found to be upregulated in pH stress and microaerobic conditions. Computational modeling, docking and simulation study suggested that DATIN might interact with TLR2. This was further confirmed through the interaction of recombinant DATIN with TLR2 expressed by HEK293 cells. When in vitro differentiated THP-1 cells were treated with recombinant DATIN, increased secretion of TNF-α, IL-1β and IL-8 was observed in a dose dependent manner. When differentiated THP-1 cells were infected with a modified BCG strain that overexpressed DATIN, augmented secretions of TNF-α, IL-1β and IL-8 were observed as compared to a reference BCG strain containing empty vector. Similarly, human PBMCs when infected with M. bovis BCG that overexpressed DATIN, upregulated secretion of proinflammatory cytokines IFN-γ, TNF-α, IL-1β and IL-8. The cytokine profiles dissected herein point to a possible role of DATIN in maintenance of latency with the help of the proinflammatory responses.

Biofilms: Survival and defense strategy for pathogens

Studies on biofilm related infections are gaining prominence owing to their involvement in majority of clinical infections. Biofilm, considered as a generic mechanism for survival used by pathogenic as well as non-pathogenic microorganisms, involves surface attachment and growth of heterogeneous cells encapsulated within a matrix. The matrix provides ecological niche where partnership of cells endows a community like behaviour that not only enables the cohort to survive local microenvironment stress but also channelizes them to evolve, disseminate and cause resurgence of infections. In this mini-review we highlight the mechanisms used by microbes to develop and sustain biofilms, including the influence of the microbiota. Several strategies to target biofilms have been validated on certain groups of microorganisms and these basically target different stages in the life cycle of biofilm, however comprehensive methods to target microbial biofilms are relatively unknown. In the backdrop of recent reports suggesting that biofilms can harbour multiple species of organisms, we need to relook and devise newer strategies against biofilms. Effective anti-biofilm strategies cannot be confined to a single methodology that can disrupt one pathway but should simultaneously target the various routes adopted by the microorganisms for survival within their ecosystem. An overview of the currently available drugs, their mode of action, genomic targets and translational therapies against biofilm related infection are discussed.

Commentary: Modification of Host Responses by Mycobacteria

1 Molecular Infection and Functional Biology Laboratory, Kusuma School of Biological Sciences, Indian Institute of Technology-Delhi, New Delhi, India, 2 School of Life Sciences, Jawaharlal Nehru University, New Delhi, India, 3 Inflammation Biology and Cell Signaling Laboratory, National Institute of Pathology, New Delhi, India, 4 Jamia Hamdard, Institute of Molecular Medicine, New Delhi, India, 5 Dr Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Hyderabad, India

Immunodominant protein MIP_05962 from Mycobacterium indicus pranii displays chaperone activity

Tuberculosis, a contagious disease of infectious origin is currently a major cause of deaths worldwide. Mycobacterium indicus pranii (MIP), a saprophytic nonpathogen and a potent immunomodulator is currently being investigated as an intervention against tuberculosis along with many other diseases with positive outcome. The apparent paradox of multiple chaperones in mycobacterial species and enigma about the cellular functions of the client proteins of these chaperones need to be explored. Chaperones are the known immunomodulators; thus, there is need to exploit the proteome of MIP for identification and characterization of putative chaperones. One of the immunogenic proteins, MIP_05962 is a member of heat shock protein (HSP) 20 family due to the presence of α‐crystallin domain, and has amino acid similarity with Mycobacterium lepraeHSP18 protein. The diverse functions of M. lepraeHSP18 in stress conditions implicate MIP_05962 as an important protein that needs to be explored. Biophysical and biochemical characterization of the said protein proved it to be a chaperone. The observations of aggregation prevention and refolding of substrate proteins in the presence of MIP_05962 along with interaction with non‐native proteins, surface hydrophobicity, formation of large oligomers, in‐vivo thermal rescue of Escherichia coli expressing MIP_05962, enhancing solubility of insoluble protein maltodextrin glucosidase (MalZ) under in‐vivo conditions, and thermal stability and reversibility confirmed MIP_05962 as a molecular chaperone.

Protein adaptations in extremophiles: An insight into extremophilic connection of mycobacterial proteome

The biological paradox about how extremophiles persist at extreme ecological conditions throws a fascinating picture of the enormous potential of a single cell to adapt to homeostatic conditions in order to propagate. Unicellular organisms face challenges from both environmental factors and the ecological niche provided by the host tissue. Although the existence of extremophiles and their physiological properties were known for a long time, availability of whole genome sequence has catapulted the study on mechanisms of adaptation and the underlying principles that have enabled these unique organisms to withstand evolutionary and environmental pressures. Comparative genomics has shown that extremophiles possess the unique set of genes and proteins that empower them with biochemical machinery necessary to thrive in extreme environments. The presence of these proteins safeguards the cell against a wide array of extreme conditions such as temperature, pressure, radiations, chemicals, drugs etc. An insight into these adaptive mechanisms in extremophiles may help us to devise strategies to alter the genes and proteins that may have therapeutic potential and commercial value. Here we present an overview of the various adaptations in extremophiles. We also try to explain how mycobacterium channelizes its proteome to survive in stress conditions posed by host immune system.

The PGRS Domain of Mycobacterium tuberculosis PE_PGRS Protein Rv0297 Is Involved in Endoplasmic Reticulum Stress-Mediated Apoptosis through Toll-Like Receptor 4

ABSTRACT The genome of Mycobacterium tuberculosis, the causal organism of tuberculosis (TB), encodes a unique protein family known as the PE/PPE/PGRS family, present exclusively in the genus Mycobacterium and nowhere else in the living kingdom, with largely unexplored functions. We describe the functional significance of the PGRS domain of Rv0297, a member of this family. In silico analyses revealed the presence of intrinsically disordered stretches and putative endoplasmic reticulum (ER) localization signals in the PGRS domain of Rv0297 (Rv0297PGRS). The PGRS domain aids in ER localization, which was shown by infecting macrophage cells with M. tuberculosis and by overexpressing the protein by transfection in macrophage cells followed by activation of the unfolded protein response, as evident from increased expression of GRP78/GRP94 and CHOP/ATF4, leading to disruption of intracellular Ca2+ homeostasis and increased nitric oxide (NO) and reactive oxygen species (ROS) production. The consequent activation of the effector caspase-8 resulted in apoptosis of macrophages, which was Toll-like receptor 4 (TLR4) dependent. Administration of recombinant Rv0297PGRS (rRv0297PGRS) also exhibited similar effects. These results implicate a hitherto-unknown role of the PGRS domain of the PE_PGRS protein family in ER stress-mediated cell death through TLR4. Since this protein is already known to be present at later stages of infection in human granulomas it points to the possibility of it being employed by M. tuberculosis for its dissemination via an apoptotic mechanism. IMPORTANCE Apoptosis is generally thought to be a defense mechanism in protecting the host against Mycobacterium tuberculosis in early stages of infection. However, apoptosis during later stages in lung granulomas may favor the bacterium in disseminating the disease. ER stress has been found to induce apoptosis in TB granulomas, in zones where apoptotic macrophages accumulate in mice and humans. In this study, we report ER stress-mediated apoptosis of host cells by the Rv0297-encoded PE_PGRS5 protein of M. tuberculosis exceptionally present in the pathogenic Mycobacterium genus. The PGRS domain of Rv0297 aids the protein in localizing to the ER and induces the unfolded protein response followed by apoptosis of macrophages. The effect of the Rv0297PGRS domain was found to be TLR4 dependent. This study presents novel insights on the strategies employed by M. tuberculosis to disseminate the disease. Apoptosis is generally thought to be a defense mechanism in protecting the host against Mycobacterium tuberculosis in early stages of infection. However, apoptosis during later stages in lung granulomas may favor the bacterium in disseminating the disease. ER stress has been found to induce apoptosis in TB granulomas, in zones where apoptotic macrophages accumulate in mice and humans. In this study, we report ER stress-mediated apoptosis of host cells by the Rv0297-encoded PE_PGRS5 protein of M. tuberculosis exceptionally present in the pathogenic Mycobacterium genus. The PGRS domain of Rv0297 aids the protein in localizing to the ER and induces the unfolded protein response followed by apoptosis of macrophages. The effect of the Rv0297PGRS domain was found to be TLR4 dependent. This study presents novel insights on the strategies employed by M. tuberculosis to disseminate the disease.

A Multi-locus Probe for Human DNA Fingerprinting Based on chi-like Sequences

DNA fingerprinting probes based on Jeffreys minisatellite core or those exhibiting similarities with this G-rich core consensus (GGGCAGGAXG), such as the (GTG)5 simple repeat (Schafer et-al 1988) or random G-rich oligodeoxynucleotides (Vergnaud 1990) presumably corresponding to VNTRs, share a degree of homology with the bacterial recombinator activator signal (chi), which is believed to serve as foci for recombination (Jarman and Wells 1988, Murray et-al 1988).