Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria

Most bacteria, including mycobacteria, generate extracellular vesicles (EVs). Since bacterial EVs (bEVs) contain a subset of cellular components, ...

NCR Biotech Science Cluster

Identifying protein-protein interactions between the machine components of bacterial secretion systems and their cognate substrates is essential. Establishing which component and substrate interactions are direct or indirect further facilitates (1) advancing the architecture and assembly of the machines and (2) understanding the substrates' translocation mechanistics. Currently, though biochemical means exist for identifying such direct interactions, they primarily remain in vitro and are quite labor intensive. Thus, adopting genetic approaches to help visualize these interactions in vivo is quick and advantageous. Here I describe bimolecular fluorescence complementation and cytology-based two-hybrid assays that could easily be adopted to understand the bacterial secretions systems.

Protein-Protein Interactions: Cytology Two-Hybrid.

The 5th Global Forum on TB Vaccines was held in New Delhi, India from 20 to 23 February 2018. This was the largest Global Forum on TB Vaccines to date with nearly 350 participants from more than 30 countries. The program included over 60 speakers in 12 special, plenary and breakout sessions and 72 posters. This Global Forum brought a great sense of momentum and excitement to the field. New vaccines are in clinical trials, new routes of delivery are being tested, novel assays and biomarker signatures are being developed, and the results from the first prevention of infection clinical trial with the H4:IC31 vaccine candidate and BCG revaccination were presented. Speakers and participants acknowledged the significant challenges that the TB vaccine R&D field continues to face - including limited funding, and the need for novel effective vaccine candidates and tools such as improved diagnostics and biomarkers to accurately predict protective efficacy. New solutions and approaches to address these challenges were discussed. The following report presents highlights from talks presented at this Global Forum. A full program, abstract book and presentations (where publicly available) from the Forum may be found at tbvaccinesforum.org.

Meeting report: 5th Global Forum on TB Vaccines, 20-23 February 2018, New Delhi India.

Sepsis is a highly heterogeneous, life-threatening organ dysfunction primarily caused by a dysregulated immune response to counter bacterial, viral, or fungal infections, resulting in haemodynamic changes and significant morbidity and mortality across all ages. In recent times, it has become one of the foremost causes of morbidity and mortality among newborns globally. The neonates, particularly the preterm neonates, due to their immature immune systems and non-canonical microbial community acquisition in the gastrointestinal tract and other body habitats, are adversely affected compared to the elderly with immunocompromised conditions. The neonates could acquire microbiota in utero or during delivery from the mother's genital tract or postnatally from contact with hospital personnel and the immediate hospital environment after the birth. Other factors that may enhance the risk include early colonization of microbiota by pathogens that trigger dysbiosis of the gut microbiome accompanied by a dysregulated immune response, organ dysfunction, and potential death. The sepsis-linked mortality could be prevented by timely diagnosis, selective antibiotic therapy, and supportive postnatal care. Infections due to antibiotic-resistant bacteria severely restrict possible therapeutic options, thus extending hospital stays. A comprehensive analysis of the infecting pathogens, cognate host responses, and the microbiota present would certainly help formulate appropriate interventions.

Gut microbiome dysbiosis in neonatal sepsis.

To survive and establish a niche for themselves, bacteria constantly evolve. Toward that, they not only insert point mutations and promote illegitimate recombinations within their genomes but also insert pieces of 'foreign' deoxyribonucleic acid, which are commonly referred to as 'genomic islands' (GEIs). The GEIs come in several forms, structures and types, often providing a fitness advantage to the harboring bacterium. In pathogenic bacteria, some GEIs may enhance virulence, thus altering disease burden, morbidity and mortality. Hence, delineating (i) the GEIs framework, (ii) their encoded functions, (iii) the triggers that help them move, (iv) the mechanisms they exploit to move among bacteria and (v) identification of their natural reservoirs will aid in superior tackling of several bacterial diseases, including sepsis. Given the vast array of comparative genomics data, in this short review, we provide an overview of the GEIs, their types and the compositions therein, especially highlighting GEIs harbored by two important pathogens, viz. Acinetobacter baumannii and Klebsiella pneumoniae, which prominently trigger sepsis in low- and middle-income countries. Our efforts help shed some light on the challenges these pathogens pose when equipped with GEIs. We hope that this review will provoke intense research into understanding GEIs, the cues that drive their mobility across bacteria and the ways and means to prevent their transfer, especially across pathogenic bacteria.

Genomic islands and their role in fitness traits of two key sepsis-causing bacterial pathogens.

Identifying protein-protein interactions between machine components of bacterial secretion systems and their cognate substrates is central to delineating how the machines operate to translocate their substrates. Further, establishing which among the machine components and their substrates interact with each other facilitates (i) advancement in our understanding of the architecture and assembly of the machines, (ii) understanding the substrates' translocation routes and mechanisms, and (iii) how the machines and the substrates talk to each other. Currently, though diverse biochemical methods exist in identifying direct and indirect protein-protein interactions, they primarily remain in vitro and can be quite labor intensive. They also may capture/exhibit false-positive interactions because of barrier breakdowns as part of methodology. Thus, adopting novel genetic approaches to help visualize the same in vivo can yield quick, advantageous, reliable, and informative protein-protein interactions data. Here, we describe the easily adoptable bimolecular fluorescence complementation and cytology-based two-hybrid assays to understand the bacterial secretions systems.

Krishnamohan Atmakuri

Bacterial Pathogenesis Laboratory,
Infectious Diseases and Immunology Group,
Translational Health Science and Technology Institute,
Bacterial Pathogenesis Laboratory, Infectious Diseases and Immunology Group, Translational Health Science and Technology Institute

NCR Biotech Science Cluster

Enrichment of Native and Recombinant Extracellular Vesicles of Mycobacteria