Cellular and molecular level host-pathogen interactions in Francisella tularensis: A microbial gene network study
Graphical Abstract
A total number of 33 whole genome sequences available for various F. tularensis strains were downloaded from NCBI database. The phylogenetic tree was constructed using ParSNP tool and visualized using iTOL tool. The representative strain F. tularensis SUH4 was highlighted in yellow color. (A) Phylogenetic tree in rooted tree layout, and (B) Circular tree view of phylogenetic tree.
Introduction
The deliberate use of biological agents solely intended to harm and cause massive deaths of humans and livestock is termed as bioterrorism. A threat of bioterrorism has now increased because of advancements made in the fields of biotechnology and biochemistry that has simplified the development of new biological warfare agents. F. tularensis is one among the known bio-warfare agents that cause severe disease in animals and also in humans (Forsman et al., 2000, Riedel, 2004). F. tularensis is a Gram-negative intracellular bacterium and is the etiological agent of the disease tularemia. Francisella belongs to the Francisellaceae family and further classified into γ subclass of proteobacteria. Phylogenetic studies on human pathogens have shown that F. tularensis is distantly related to Coxiella burnetii and Legionella spp. (Larsson et al., 2005). The infection caused by F. tularensis can be acquired through various infectious routes such as inhalation, ingestion, percutaneous or direct eye infection (Brett Moreau and Mann, 2013). Humans can become infected with F. tularensis in a number of ways, including arthropod bites, handling sick animal tissues or fluids, direct contact with or ingestion of contaminated water, food, or soil, and breathing infective aerosols. Studies on human volunteers indicate tularemia can be deadly in up to 30% of untreated cases, with the death rate approaching 90% in pneumonic infections (El-Etr et al., 2009).
The strains of F. tularensis family are further divided into two subspecies namely type-A and type-B. The type-A (F. tularensis subsp. tularensis) includes more severe forms of disease which can cause infection even at low dose, whereas type-B (F. tularensis subsp. holarctica) is comparatively less infectious than the type-A subspecies (Larson et al., 2011, Santiago et al., 2015). Rabbits, ticks, and sheep are associated with transmission of F. tularensis subspecies tularensis, whereas F. tularensis subspecies holarctica is typically isolated from streams, ponds, lakes, and rivers (WHO, 2007). Due to its exceptional characteristics such as the virulent nature, high mortality rate, easy aerosolization and low infectious dose, F. tularensis strains were classified as Tier 1 agents and the usage of these strains were reported as bioweapons. As per the reports, F. tularemia was first believed to be used as a bioweapon during 1320–1318 BCE (Gürcan, 2014). Due to the re-emergence of tularemia and high number of new cases globally, it gained the interest of scientific community in the recent years. Francisella subspecies have been discovered all across the world, with varied geographic distributions and disease potential. In the period between 2001 and 2010, Kosovo had the highest annual incidence rate in Europe, at 5.2 per 100,000. Sweden, Finland, Slovakia, Czech Republic, Norway, Serbia-Montenegro, Hungary, Bulgaria, and Croatia follow with rates of 2.80, 1.19, 1.0, 0.81, 0.42, 0.4, 0.36, 0.21, and 0.15 per 100,000 population respectively (Gürcan, 2014, Stidham et al., 2018).
F. tularensis is an intracellular pathogen, and have the capability to replicate within the macrophages. When the pathogen enters the host body the macrophages engulfs them through a distinctive form of phagocytosis called looping phagocytosis. In this process the macrophage engulfs a large space around the bacterium in actin dependent manner. Interestingly, the bacterium has acquired a survival strategy to escape phagocytosis by restricting phagosome acidification and thus prevents the maturation, and hence the bacterium escapes the phagocytosis and enters into the cytoplasm (Steiner et al., 2014). The growth of bacterium in intracellular environment purely depends on the available nutrients in host and the ability of the bacterium to utilize the nutrients for its metabolism. Therefore, one of the important keys to understand the pathogenicity of F. tularensis is by unlocking the secrets of its survival strategies inside the host cells. Host-pathogen protein-protein interactions (PPI) provide information that can help the scientists and researchers understand the disease pathogenesis, the biology of one or many molecular mechanisms, as well as the biology of the host. The recent advances in high-throughput protein interaction detection methods have led to the generation of large-scale interspecies PPI data of pathogen-human systems. Other important approach to understand the host-pathogen interactome is to find the interconnection of metabolic pathways of the host and the pathogen (Jones et al., 2014, Raghunathan et al., 2010, Solbakken et al., 2019).
For our present study, we have selected F. tularensis SCHU S4 strain, a representative strain of F. tularensis which is considered as the most virulent prototypic strain (Molins et al., 2014). We have constructed a phylogenetic tree to understand the evolutionary relationship of the representative strain SCHU S4, with other prototypes of F. tularensis. We have further collected the important proteins which were reported to have involved in interaction with the host proteins. We have used gene network approach to construct interaction networks of pathogen PPI, host PPI and host pathogen interaction (HPI) network, thus to understand the importance of these interactions at molecular level. Interaction network analysis is one of the emerging approaches to understand the molecular level interactions between the host and the pathogen (Miryala et al., 2018). Understanding the PPI in host and the pathogen will help to better understand the key pathways involved in bacterial survival and the pathways in host to combat against the bacterial infection. By studying the strategies of host and the pathogen, researchers can design a plan of action to control the pathogen’s virulence and also by boosting the host immune responses (Basu et al., 2021, Parvati Sai Arun et al., 2018, Sevimoglu and Arga, 2014). Previously we have studied the gene interaction analysis to understand the drug resistance patterns displayed by the pathogenic bacteria (Ashok et al., 2021, Debroy et al., 2020, Miryala et al., 2021, Miryala et al., 2019a, Miryala et al., 2019b; Miryala et al., 2020a, Miryala et al., 2020b; Miryala and Ramaiah, 2018; Naha et al., 2020). In the present study we have made an effort to understand the molecular level interactions between the human and bacterial proteins. The functional enrichment analysis revealed the important pathways which play significant roles in pathogen survival and also in host defense mechanisms. The genes predicted as the potential drug targets might help us in developing successful treatment strategies and to formulate new antibiotics to treat tularemia.
Section snippets
NCBI-Genome database
The Genome database (https://www.ncbi.nlm.nih.gov/genome) in National Center for Biotechnology Information (NCBI) and it consists of resources which include the information on large scale genomics projects, genome sequences and genome assemblies. It also includes the mapped annotations such as variations, markers and epigenomics studies related data (Acland et al., 2013, G-Preciado et al., 2009).
ParSNP
ParSNP (https://github.com/marbl/parsnp) is a tool designed to align the genome sequences rapidly.
Results and discussions
F. tularensis SCHU S4 strain is considered as the representative strain of F. tularensis. There are a total of 264 genome entries available for F. tularensis; from the list we have further collected the sequences, for which the genome assembly status is updated (Supplementary file S1). A total of 33 whole genome sequences, which included both type A and type B strains were used for the construction and analysis of the phylogenetic tree (Table 1). We have further constructed the gene interaction
Conclusion
F. tularensis interactions in humans have a high mortality rate. Francisella species exploits various complex defensive mechanisms to invade the host immune responses. In our present study, we have made an attempt to understand the gene interaction networks of host genes and pathogen genes and also the functions associated with these interacting genes. The interaction network has shown dense interactions between the host and pathogen genes along with the functional partners. The functional
Funding information
Sudha Ramaiah gratefully acknowledges the Indian Council of Medical Research (ICMR), the Government of India agency for the research grant (IRIS ID: 2020–0690). Sravan Kumar Miryala heartfully thanks the ICMR, for the senior research fellowship grant (IRIS ID: 2020–7788).
CRediT authorship contribution statement
Sudha Ramaiah: Conceptualization, Methodology, Supervision. Sravan Kumar Miryala: Data curation, Analysis, Visualization, Writing − original draft preparation.
Conflict of interest statement
The authors declare that there is no conflict of interest.
Acknowledgments
The authors would like to thank Dr. Anand Anbarasu for his constant guidance during the entire study. The authors would like to thank the management of Vellore Institute of Technology (VIT), Vellore for providing the necessary facilities to carry out this research work.
Author contributions
SR designed the study; MSK collected the data and carried out the computation and generated figures; SR and MSK analyzed the data and wrote the manuscript.
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