Neha Dhasmana

Mycobacterium tuberculosis Cyclophilin A Uses Novel Signal Sequence for Secretion and Mimics Eukaryotic Cyclophilins for Interaction with Host Protein Repertoire

Cyclophilins are prolyl isomerases with multitude of functions in different cellular processes and pathological conditions. Cyclophilin A (PpiA) of Mycobacterium tuberculosis is secreted during infection in intraphagosomal niche. However, our understanding about the evolutionary origin, secretory mechanism or the interactome of M. tuberculosis PpiA is limited. This study demonstrates through phylogenetic and structural analyses that PpiA has more proximity to human cyclophilins than the prokaryotic counterparts. We report a unique N-terminal sequence (MADCDSVTNSP) present in pathogenic mycobacterial PpiA and absent in non-pathogenic strains. This sequence stretch was shown to be essential for PpiA secretion. The overexpression of full-length PpiA from M. tuberculosis in non-pathogenic Mycobacterium smegmatis resulted in PpiA secretion while truncation of the N-terminal stretch obstructed the secretion. In addition, presence of an ESX pathway substrate motif in M. tuberculosis PpiA suggested possible involvement of Type VII secretion system. Site-directed mutagenesis of key residues in this motif in full-length PpiA also hindered the secretion in M. smegmatis. Bacterial two-hybrid screens with human lung cDNA library as target were utilized to identify interaction partners of PpiA from host repertoire, and a number of substrates with functional representation in iron storage, signal transduction and immune responses were detected. The extensive host interactome coupled with the sequence and structural similarity to human cyclophilins is strongly suggestive of PpiA being deployed by M. tuberculosis as an effector mimic against the host cyclophilins.

Ser/Thr protein kinase PrkC-mediated regulation of GroEL is critical for biofilm formation in Bacillus anthracis

PrkC is a conserved Ser/Thr protein kinase encoded in Bacillus anthracis genome. PrkC is shown to be important for B. anthracis pathogenesis, but little is known about its other functions and phosphorylated substrates. Systemic analyses indicate the compelling role of PrkC in phosphorylating multiple substrates, including the essential chaperone GroEL. Through mass spectrometry, we identified that PrkC phosphorylates GroEL on six threonine residues that are distributed in three canonical regions. Phosphorylation facilitates the oligomerization of GroEL to the physiologically active tetradecameric state and increases its affinity toward the co-chaperone GroES. Deletion of prkC in B. anthracis abrogates its ability to form biofilm. Overexpression of native GroEL recovers the biofilm-forming ability of prkC deletion strain. Similar overexpression of GroEL phosphorylation site mutants (Thr to Ala) does not augment biofilm formation. Further analyses indicate the phosphorylation of GroEL in diverse bacterial species. Thus, our results suggest that PrkC regulates biofilm formation by modulating the GroEL activity in a phosphorylation-dependent manner. The study deciphers the molecular signaling events that are important for biofilm formation in B. anthracis.Anthrax bacteria: a step in the pathway to biofilmsAn enzyme that adds phosphate groups to other proteins, PrkC, mediates molecular signaling events that allow anthrax bacteria to form biofilms. Bacillus anthracis is widely used as a model to explore the formation of biofilms that allows many bacterial infections to resist immune defenses. An international research team led by Yogendra Singh and Andaleeb Sajid at the CSIR-Institute of Genomics and Integrative Biology in Delhi, India, studied the bacterial protein kinase PrkC. The researchers found that PrkC phosphorylates a “chaperone” protein that assist the assembly and disassembly of other protein-based structures. This signaling protein and the chaperone help in biofilm formation. Establishing this link in the signaling chain leading to biofilms will guide future research to combat the role of biofilms in disease.

Bacterial Virulence Factors: Secreted for Survival

Abstract Virulence is described as an ability of an organism to infect the host and cause a disease. Virulence factors are the molecules that assist the bacterium colonize the host at the cellular level. These factors are either secretory, membrane associated or cytosolic in nature. The cytosolic factors facilitate the bacterium to undergo quick adaptive—metabolic, physiological and morphological shifts. The membrane associated virulence factors aid the bacterium in adhesion and evasion of the host cell. The secretory factors are important components of bacterial armoury which help the bacterium wade through the innate and adaptive immune response mounted within the host. In extracellular pathogens, the secretory virulence factors act synergistically to kill the host cells. In this review, we revisit the role of some of the secreted virulence factors of two human pathogens: Mycobacterium tuberculosis—an intracellular pathogen and Bacillus anthracis—an extracellular pathogen. The advances in research on the role of secretory factors of these pathogens during infection are discussed.

clpC operon regulates cell architecture and sporulation in Bacillus anthracis

The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.

Combating Staphylococcal Infections Through Quorum Sensing Inhibitors

Staphylococcus aureus is a clinically important pathogen mainly causing hospital borne infections. These bacterial infections range from mild skin infections to serious health threats like endocarditis, osteomyelitis, and pneumonia. Few strains have developed resistance against antibiotics used to treat S. aureus infections and are termed as Methicillin Resistant S. aureus strains. The pathogen releases Auto Inducing Peptides to establish cell density dependent inter-cell communication, also known as quorum sensing (QS). QS results in the expression of accessory gene regulator system. It causes successful biofilm formation and enhanced expression of toxins. QS mediated biofilm formation provides an additional resistance against the antibiotics used. An innovative therapeutic approach has been studied vastly in last decade to deal with severe infections using specific QS inhibitors (QSIs). This chapter comprehensively describes the QSIs studied to control the infections caused by S. aureus strains.

Quorum-Sensing Systems in Bacillus

Since centuries bacteria were thought to be unicellular organisms. The discovery of bacterial communication through small molecules has asserted that bacteria can efficiently coordinate intraspecies as well as interspecies. The bacteria become more benefitted and suitable of behaving like a multicellular organism to adopt new modes of growth in limited nutrient supply. Under adverse conditions, single bacterial cell has less chance to survive in isolation; consequently bacterial language has been developed during evolution to communicate with its neighbours through self-generated signals (Bassler and Losick 2006). These signalling small molecules are called as pheromones or autoinducers. These autoinducers sense a critical bacterial density in population (Kievit et al. 2000; Williams et al. 2007).

Microbial Vesicles: From Ecosystem to Diseases

The production of outer membrane vesicles (OMVs) is conserved in eukaryotes and prokaryotes. OMVs are double-layered structures with contents from outer membrane, periplasmic space, and even cytosol. Some of them have been shown to contain nucleic acids as well, which explains the specialized system for the packaging of these vesicles. OMVs mediate essential processes such as transport of nutrients, antigens, and virulence factors, etc., which help the microorganisms in communication as well as in killing of other microbial cells. The biogenesis of OMVs differs in bacteria and archaea. The archaeal OMV biogenesis is similar to eukaryotes involving ESCRT machinery, while in gram-negative bacteria, it occurs either due to broken OM-PG interaction or due to increased periplasmic pressure. OMVs containing antigens have been recently explored for use as vaccine which provides another dimension for its applications.

Sporulation, a Pitfall in the Path of PHB Production

The concept of bioplastic is fascinating to our world, because of its potentiality to deal with one of the major global problems like plastic pollution (Kalia et al. J Sci Ind Res 59:433–445, 2000; Kalia et al. Nat Biotechnol 21:845–846, 2003). Polyhydroxybutyrates (PHB) are the best example for the polymers by plant or microorganisms from a wide range of habitats (Reddy et al. Bioresour Technol 87:137–146, 2003; Porwal et al. Bioresour Technol 99:5444–5451, 2008; Singh. Environ Microbiol 17:854–864, 2015). PHB refers to the polyesters of 3-hydroxybutyrate and can be extracted from various species like Ralstonia, Bacillus, Streptomyces, Pseudomonas, etc., which are extensively discussed in published reviews (Singh et al. Microb Cell Fact 8:38, 2009; Jendrossek and Pfeiffer. Environ Microbiol 16:2357–2373, 2014). Bioplastic has the distinct feature of being biodegradable. Further, the use of biowaste as substratum for bioplastic-producing organisms presents an interesting concept to deal with another global problem of waste management (Kumar et al. J Appl Microbiol 106:2017–2023, 2009; Kumar et al. Indian J Microbiol 55:1–7, 2015; Kumar et al. Int J Biol Macromol, 2015; Patel et al. Biomas Bioenerg 36:218–225, 2012; Patel et al. Bioresour Technol 176:136–141, 2015). Both Gram-positive and Gram-negative bacteria are reported to produce polyhydroxyalkanoates (PHA); among them Gram-negative bacteria, Ralstonia eutropha is the most extensively studied organism (Brigham et al. Appl Environ Microbiol 78:8033–8044, 2012). One major rationale to investigate the Gram-positive bacteria for their ability to produce PHB is the absence of immunogenic lipopolysaccharide which co-purifies with the PHB when Gram-negative organisms are employed, making PHB non-appealing for the use in medical purposes like various human tissue grafts (Valappil et al. Antonie Van Leeuwenhoek 91:1–17, 2007; Singh et al. Microb Cell Fact 8:38, 2009). Additional appeal for using Gram-positive bacteria, specifically Bacillus spp., is its ability to produce copolymers which are superior to their counterparts, that is, homopolymers given their enhanced characteristics like elasticity, etc. (Patel et al. Indian J Microbiol 51:418–423, 2011; Kumar et al. Indian J Microbiol 54:151–157, 2014; Kumar et al. Int J Biol Macromol, 2015). Gram-positive bacteria exist in two alternative phases in its life cycle, that is, vegetative cells and sporulation. The adverse environmental conditions drive the Bacillus vegetative cells into their transition to spores. Sporulation is an intrinsic characteristic of the Bacillus species and is mainly regulated by a master regulator of sporulation, namely, Spo0A (Slepecky and Law. J Bacteriol 82:37–42, 1961; Singh et al. Indian J Microbiol 55:234, 2015).

Frontiers in Biomedical Engineering: PHA-Fabricated Implants

Polyhydroxyalkonoates (PHAs) are biological in origin, organic polyesters comprising the industrial and biomedical interest. This chapter summarizes the current advances, applications, limitations, and challenges of biopolymers in medicine. Biopolymers not only substitute the existing polymers, but novel combinations of diverse PHAs broaden the applicability and utility. The PHA-based implants are new dimensions of future in biomedical engineering.