Education and training:

Dr. Girish K. Radhakrishnan obtained MSc in Microbiology (1999) from School of Biosciences, Mahatma Gandhi University, Kerala and PhD in Biotechnology (2006) from School of Biotechnology, Madurai Kamaraj University, Tamil Nadu. He did his postdoctoral research (2006-2010) at the University of Wisconsin-Madison, USA and worked as Research Assistant Professor at the same university (2010-2012). He joined NIAB on 03 December 2012.

We are studying various aspects of the infectious intracellular bacterial pathogen Brucella, causing the world-wide zoonotic disease, brucellosis. Brucellosis poses a serious veterinary and public health problem in India and the annual loss in the county is estimated to be 28,300 cores. There is no human vaccine for brucellosis and the available animal vaccines have many disadvantages. The treatment of brucellosis with antibiotics is inefficient due to frequent treatment failures, prolonged duration of treatment with multiple antibiotics and occurrence of relapse. The diagnosis of brucellosis is challenging due to its atypical symptoms and poor sensitivity and specificity of available serodiagnostic assays. Compared to other bacterial pathogens, minimal information is available on the virulence mechanisms that enable Brucella to survive and replicate in the host. Our studies focus on understanding the virulence attributes of Brucella that enable this bacterial pathogen to evade/suppress host innate and adaptive immunity for its chronic persistence. We employ various genomic and proteomic techniques including protein micro-arrays, CRISPR-Cas9 system, siRNA, lentivirus/AAV-based gene delivery, functional genomic yeast screening and infection studies using gene KO mice. Our ultimate goal is to develop improved vaccines, therapeutics and diagnostic assays for animal and human brucellosis. Aberrant activation of innate immune signaling leads to various inflammatory and infectious diseases. We use virulence proteins of Brucella as the tool to study the regulation of innate immune signaling with the objective of developing novel therapeutics for inflammatory disorders. Some of the research highlights are mentioned below:-

Identification and characterization of immunodominant proteins of Brucella.

Identification and characterization of immunodominant antigens that are secreted or shed by Brucella in the blood of infected host can provide valuable insights into the virulence properties of the pathogen. This information can be used for developing improved sero-diagnostic assays and next generation vaccines for animal and human brucellosis. To identify the novel immunodominant antigens of Brucella, we performed a high-throughput immunoprobing of the Brucella melitensis protein micro-array with the serum samples from Brucella-infected or S19 vaccinated cattle, goat, dog and human (Fig. 1). The analysis identified the immunodominant antigens that are shared or uniquely present in various host species. Further, we developed sero-diagnostic assays based on one of the identified proteins for detection of brucellosis in animals and humans with high specificity and sensitivity (patent filed). The detailed characterization of identified immunodominant protein antigens is in progress.

Figure 1. Probing of Brucella melitensis protein microarray with brucellosis positive or negative serum sample.

The Brucella virulence protein, TcpB recruits the Cytoplasmic Linker Protein-170 (CLIP170) for attenuating the TLR2/4 signaling.

Toll-like receptors (TLRs) are crucial components of the innate immune system that recognize conserved microbial components and trigger anti-microbial responses. Brucella secretes the virulence protein, TcpB, which suppresses host innate immune responses through negative regulation of TLR2 and 4 signaling. However, the mechanism by which TcpB subverts the TLR2/4 signaling remains obscure. We performed a high-throughput yeast-two hybrid screening that identified interaction between TcpB and the host protein, CLIP170. Further studies revealed that CLIP170 attenuates TLR2/4 signaling by targeted ubiquitination and degradation of the TLR2/4 adaptor protein, TIRAP (Journal of Immunology, 2018). The neurosteroid, pregnenolone is reported to bind and activate CLIP170. Pregnenolone has been in use as an anti-inflammatory agent and as a steroid for well-being for several decades. However, the mechanism by which pregnenolone exerts its anti-inflammatory property remains unknown. Our studies revealed that pregnenolone induces ubiquitination and degradation of key proteins in the TLR signaling pathway through the activation of CLIP170 (Journal of Biological Chemistry, 2019).

The Brucella protein, TcpB induces degradation of inflammatory caspases and thereby subverts non-canonical inflammasome activation in macrophages.

The inflammasome contains intracellular receptors that recognize various pathogen-associated molecular patterns and play crucial roles in innate immune responses to invading pathogens. Non-canonical inflammasome activation is mediated by caspase-4/11, which recognizes intracellular LPS and promotes pyroptosis and secretion of proinflammatory cytokines. TcpB attenuated non-canonical inflammasome activation and suppressed pyroptosis and secretion of IL-1α and β induced by intracellular LPS delivery or infection with Brucella or Salmonella (Fig. 2). Further, we found that TcpB induced ubiquitination and degradation of the inflammatory caspases 1, 4, and 11 to attenuate non-canonical inflammasome-mediated signaling (Journal of Biological Chemistry, 2019).

Figure 2. TcpB treated cells resist pyroptotic cell death induced by LPS. Primed RAW264 macrophages were treated with MBP-TcpB or MBP alone followed by LPS transfections. Cells were then stained with Zombie Red dye to visualize pyroptotic cell death. Cells treated with TcpB excluded the dye and displayed peripheral labeling compared to MBP treated cells, which had taken up the dye and appeared bright. Images are representative of three independent experiments. Scale bar 30 µm.

High-throughput screening using siRNA or CRISPR-Cas9 to identify novel host factors that support the invasion and intracellular replication of Brucella.

We performed a high-throughput siRNA screening that identified several host proteins, which facilitate or block the invasion and intracellular replication of Brucella in macrophages. Further studies are in progress to characterize these proteins using various genomics and proteomics techniques.

1. Varadendra Mazumdar, Kiranmai Joshi, Binita Roy Nandi, Swapna Namani, Vivek Kumar Gupta and Girish Radhakrishnan (2022). Host F-Box Protein 22 Enhances the Uptake of Brucella by Macrophages and Drives a Sustained Release of Proinflammatory Cytokines through Degradation of the Anti-Inflammatory Effector Proteins of Brucella. Infection and Immunity 90(5):e0006022. doi: 10.1128/iai.00060-22. https://pubmed.ncbi.nlm.nih.gov/35420446/

2. Subathra Murugan, Padmaja Jakka, Swapna Namani,Varadendra Mujumdar and Girish Radhakrishnan (2019). The neurosteroid, pregnenolone promotes degradation of key proteins in the innate immune signalling to suppress Journal of Biological Chemistry 294 (12) 4596-4607.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433066/

3. Padmaja Jakka, Swapna Namani, Subathra Murugan, Nivedita Rai and Girish Radhakrishnan* (2018). The Brucella effector protein TcpB induces degradation of inflammatory caspases and thereby subverts non-canonical inflammasome activation in Journal of Biological Chemistry 292 (50), 20613-20627.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733597/

4. Padmaja Jakka, Bindu Bhargavi, Swapna Namani, Subathra Murugan, Gary Splitter and Girish Radhakrishnan* (2017). Cytoplasmic Linker Protein CLIP170 Negatively Regulates TLR4 Signaling by Targeting the TLR Adaptor Protein Journal of Immunology 200 (2) 704-714.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760445/

5. Sarwar Azam, Sashi Bhushan Rao, Padmaja Jakka, Veera NarasimhaRao, Bindu Bhargavi, Vivek Kumar Gupta, and Girish Radhakrishnan (2016). Genetic Characterization and Comparative Genome Analysis of Brucella melitensis Isolates from International Journal of Genomics. Volume 2016 (2016),13 pages.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976149/

6. Splitter G, Harms J, Petersen E, Magnani D, Durward M, Rajashekara G, Radhakrishnan (2014) Studying host-pathogen interaction events in living mice visualized in real time using biophotonic imaging. Methods Mol Biol. 2014;1197:67-85.https://pubmed.ncbi.nlm.nih.gov/25172275/

7. Sashi Bhushan Rao, Vivek Gupta,Mukesh Kumar, Nagendra R. Hegde, Gary A. Splitter, Pallu Reddanna and Girish K. Radhakrishnan* (2014). Draft Genome Sequence of the Field Isolate Brucella melitensis strain BM IND-1 from India. Genome Announcements 2(3):e00497-14. doi:10.1128/genomeA.00497-14.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038885/

8. Smith, A. Magnani. D, Kahn. M , Harms. J, Durward. M, Radhakrishnan. G, Liu, Y- P and Splitter, G (2013). Brucella Induces an Unfolded Protein Response via TcpB that Supports Intracellular Replication in Macrophages. PLoS Pathogen, 9(12): e1003785. doi:10.1371/journal.ppat.1003785.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3855547/

9. Radhakrishnan, G. and Splitter, G. (2012) Modulation of host microtubule dynamics by pathogenic bacteria. Biomolecular Concepts. 3 (6), 571–580.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3625037/

10. Gupta VK, Radhakrishnan G, Harms J, Splitter G. (2012) Invasive Escherichia coli vaccines expressing Brucella melitensis outer membrane proteins 31 or 16 or periplasmic protein BP26 confer protection in mice challenged with B. melitensis. Vaccine, 30, 4017- 4022.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3361596/

11. Durward M, Radhakrishnan G, Harms J, Bareiss C, Magnani D and Splitter, G. A (2012) Active Evasion of CTL Mediated Killing and Low Quality Responding CD8+ T Cells Contribute to Persistence of Brucellosis. PLoS ONE 7(4): e34925.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3338818/

12. Radhakrishnan, G, Harms, J and Splitter, G (2011). Modulation of microtubule dynamics by a TIR domain containing protein from an intracellular pathogen Brucella. Biochemical Journal 439 (1) 79-83.https://pubmed.ncbi.nlm.nih.gov/21692747/

13. Radhakrishnan, G and Splitter, G. (2010).Biochemical and functional analysis of TIR domain-containing protein from Brucella melitensis. Biochemical and Biophysical Research Communications 397(1) 59-63.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2900483/

14. Radhakrishnan, G. K., Yu, Q., Harms, J. S., Splitter, G. A. (2009). Brucella TIR domain- containing protein mimics properties of the toll-like receptor adaptor protein TIRAP. Journal of Biological Chemistry 284(15): 9892-9898.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2665112/

15. Subathra Murugan, Padmaja Jakka, Swapna Namani,Varadendra Mujumdar and Girish Radhakrishnan (2019). The neurosteroid, pregnenolone promotes degradation of key proteins in the innate immune signalling to suppress inflammation. Journal of Biological Chemistry 294 (12) 4596-4607.

16. Padmaja Jakka, Swapna Namani, Subathra Murugan, Nivedita Rai and Girish Radhakrishnan* (2018). The Brucella effector protein TcpB induces degradation of inflammatory caspases and thereby subverts non-canonical inflammasome activation in macrophages. Journal of Biological Chemistry 292 (50), 20613-20627

17. Padmaja Jakka, Bindu Bhargavi, Swapna Namani, Subathra Murugan, Gary Splitter and Girish Radhakrishnan* (2017). Cytoplasmic Linker Protein CLIP170 Negatively Regulates TLR4 Signaling by Targeting the TLR Adaptor Protein TIRAP. Journal of Immunology 200 (2) 704-714.

18. Sarwar Azam, Sashi Bhushan Rao, Padmaja Jakka, Veera NarasimhaRao, Bindu Bhargavi, Vivek Kumar Gupta, and Girish Radhakrishnan (2016). Genetic Characterization and Comparative Genome Analysis of Brucella melitensis Isolates from India. International Journal of Genomics. Volume 2016 (2016),13 pages

19. Sashi Bhushan Rao, Vivek K. Gupta,Mukesh Kumar, Nagendra R. Hegde, Gary A. Splitter, Pallu Reddanna and Girish K. Radhakrishnan (2014). Draft Genome Sequence of the Field Isolate Brucella melitensis strain BM IND-1 from India. Genome Announcements 2(3):e00497-14. doi:10.1128/genomeA.00497-14.

20. Smith, J. A. Magnani. D, Kahn. M , Harms. J, Durward. M, Radhakrishnan. G, Liu, Y-P and Splitter, G (2013).Brucella Induces an Unfolded Protein Response via TcpB that Supports Intracellular Replication in Macrophages. PLoS Pathogen, 9(12): e1003785. doi:10.1371/journal.ppat.1003785.

PhD Students:

Ms. Prachita Nandini

Mr. Varadendra Mazumdar

Ms. Sushreerekha Mallik

Ms. Binita Roy

Ms. Kiranmai Josh

Project Associates:

Deepak Kumar

Lab Alumni:

Dr. Padmaja Jakka, Post Doc, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, USA

Ongoing extramural research grants:

1. Understanding the role of an Ubiquitin Specific Peptidase in the invasion and intracellular replication of the zoonotic bacterial pathogen, Brucella
Funding agency: DBT
Duration: February 2022 to February 2025

2. Studies on the immunodominant proteins of the zoonotic pathogen, Brucella to develop improved diagnostic assays and vaccines for brucellosis.
Funding agency: DBT
Duration: September 2021 to September 2024

3. Development, evaluation and validation of peptide-based iELISA for the zoonotic diseases, (1) Brucellosis and (2) Coxiellosis (Q-fever).
Funding agency: DBT-NER
Duration: March 2021 to March 2023

Completed extramural research grants:

1. To understand the role of Cytoplasmic linker protein-170 in the down-regulation of TLR4 signaling. Funding agency: DBT.

2. Understanding the mechanism of host innate immune suppression by the Brucella effector protein, TcpB to identify novel drug targets for brucellosis. Funding agency: DST-SERB.

3. Development of peptide-based anti-inflammatory drug for septicemia. Funding agency: DST-DPRP.

4. To develop novel therapeutics for brucellosis: Identification and characterization of host factors supporting Brucella replication. Funding agency: DBT.

5. Understanding the immune mechanism of host disease resistance and development of marker vaccines and DIVA tests for Peste des Petits Ruminants (PPR). Funded by DBT-BBSRC (multi-institutional project). Funding agency: DBT-BBSRC.

Laboratory of Immunology and Microbial Pathogenesis
National Institute of Animal Biotechnology (NIAB),
Opp. Journalist Colony, Near Gowlidoddi,
Extended Q City Road, Gachibowli,
Hyderabad, Telangana-500 032,
India.

Email: girish[at]niab[dot]org[dot]in
Tel: +91-(0)40-2312-0142

Development of “Differentiating Infected from Vaccinated Animals (DIVA)” capable serodiagnostic assays for brucellosis:
Early detection and control of brucellosis in livestock is crucial for controlling the disease in humans. Existing serodiagnostic assays for brucellosis have many disadvantages such as poor sensitivity/specificity and lack of DIVA capability. We developed an indirect ELISA using an immunodominant protein (BM5) from Brucella for serodiagnosis of brucellosis in animals and humans. The BM5-ELISA can detect anti-Brucella antibodies in the Brucella-infected animals and humans with high specificity and sensitivity. The BM5-ELISA can efficiently differentiate Brucella abortus S19-vaccinated from naturally infected cattle. We developed recombinant BM5 protein as well as peptide (14 amino acids)-based ELISAs.

Patent applications filed:-

1) Cell permeable peptides; Indian Patent application no. 202141048139

2) Novel anti-inflammatory peptides; Indian Patent application no. 202141048130

3) Immunodominant protein and peptide-based brucellosis diagnosis kits and devices to differentiate infected animals from Brucella abortus S19-vaccinated animals. Indian Patent Application No. 201941010993

Technology Transferred:-

BM5 protein/peptide-based brucellosis diagnostic assays with DIVA capability

Company: Engrave Bio Labs, Hyderabad

Web links
Pubmed: https://www.ncbi.nlm.nih.gov/pubmed/?term=Girish+Radhakrishnan
Google Scholar Link: https://scholar.google.com/citations?user=IEFTDcAAAAAJ&hl=en

Postdoctoral Research Opportunities are available.