Pan-proteome profiling of emerging and re-emerging zoonotic pathogen Orientia tsutsugamushi for getting insight into microbial pathogenesis

Highlights

• Scrub typhus is neglected zoonotic disease which infects more than one million people across the globe.

• Pan-proteome of Orientia tsutsugamushi (Ott) is estimated to be extensive in nature and rich in core proteins.

• The analysis of Ott pan-proteome helps in the identification of potential therapeutic targets.

• The predicted proteins envisioned as potential candidates for designing antimicrobial agents to combat scrub typhus.

Abstract

With the occurrence and evolution of antibiotic and multidrug resistance in bacteria most of the existing remedies are becoming ineffective. The pan-proteome exploration of the bacterial pathogens helps to identify the wide spectrum therapeutic targets which will be effective against all strains in a species. The current study is focused on the pan-proteome profiling of zoonotic pathogen Orientia tsutsugamushi (Ott) for the identification of potential therapeutic targets. The pan-proteome of Ott is estimated to be extensive in nature that has 1429 protein clusters, out of which 694 were core, 391 were accessory, and 344 were unique. It was revealed that 622 proteins were essential, 222 proteins were virulent factors, and 42 proteins were involved in antibiotic resistance. The potential therapeutic targets were further classified into eleven broad classes among which gene expression and regulation, transport, and metabolism were dominant. The biological interactome analysis of therapeutic targets revealed that an ample amount of interactions were present among the proteins involved in DNA replication, ribosome assembly, cellwall metabolism, cell division, and antimicrobial resistance. The predicted therapeutic targets from the pan-proteome of Ott are involved in various biological processes, virulence, and antibiotic resistance; hence envisioned as potential candidates for drug discovery to combat scrub typhus.

Introduction

The acute febrile zoonotic disease “scrub typhus” is caused by the intracellular bacterium Orientia tsutsugamushi (Ott), which threatens about one billion and infect nearly one million people across the globe [1,2]. Orientia tsutsugamushi is an obligate intracellular gram-negative bacterium that belongs to order Rickettsiales and family Rickettsiaceae. The species name of the bacterium is obtained from the Japanese word “tsutsuga” meaning “disease” and “mushi” means “mite” [3]. The pathogenic bacterium is transmitted to humans by feeding larvae of trombiculid mites (genus Leptotrombidium) and mite also serves as the reservoir of Ott [4]. The microorganism spreads through blood and lymphatic fluid, causing symptoms like rash, fever, and elevated levels of liver enzymes and C-reactive protein [5].

Scrub typhus is one of the oldest vector-borne diseases endemic to the “tsutsugamushi triangle” in Asia-Pacific. The tsutsugamushi triangle covers about eight million sq km area including Northern Australia, Northern Japan, Taiwan, Russia, China, Philippines, South Korea, India, Nepal, and Afghanistan [6]. The disease is endemic in the tsutsugamushi triangle but cases also reported from South America, Middle East, Africa, and Europe, shows the wider geographical distribution of the Orientia genus [5,7]. Furthermore, scrub typhus is considered a neglected zoonotic disease but has an ever-widening impact throughout the world.

Scrub typhus is an endemic and emerging and re-emerging infectious disease in India. During the Second World War scrub typhus broke out in an epidemic form in West Bengal and Assam states of India [8]. The disease can simultaneously lead to various other health complications, i.e., neurological, respiratory, hematological, and cardiac problems [9]. Moreover, scrub typhus is the major cause of acute encephalitis syndrome among Indian children [10].

Centre for Disease Control and Prevention (CDC) mentioned that no satisfactory vaccine had been developed yet for scrub typhus. There are some antibiotics currently available to treat scrub typhus, though; uncertain evidences of antibiotic resistance have been documented [[11], [12], [13]]. The present need is to design effective control measures to eradicate scrub typhus due to its non-specific representation and high mortality rate [3].

Closely related bacterial species share large number of genes; however, some variable genes concentrate bacterial species gene pool due to genetic diversity and further expand species pan-genome [14,15]. The notion of bacterial pan-genome was established about 15 years back and can be defined as the entire genic content of all strains in a species [[16], [17], [18]]. The three components of pan-genome are: (i) core genome composed of genes present in all strains, (ii) accessory genome consisting set of genes absent from one or more strains, and (iii) unique genome confined to individual strain [[16], [17], [18]].

Scientists used a single reference genome of each species in the early period of the genomic era for various genetic analyses [15,19]. The development and expansion of next generation sequencing (NGS) technologies broaden our perceptive on microbial genetics, physiology, and functional diversity at the metagenomic echelon which makes realization to the scientific community that it is not sufficient to extract all the genetic information from a single reference genome [14,15,17]. The large datasets provided by these sequencing technologies further assist in-depth analysis of intra-species comparisons and diversity from various prospects like understanding the genetic variations in and among pathogenic groups and designing therapeutic targets [15,17,20]. Although, genetic diversity is measurable at genetic level, yet, the best impact is obtained by analyzing “protein”, i.e., functional unit of the cell [21]. Therefore, the use of proteomics approaches is offered as one of the most favorable or convenient way to get insight into the complex functional diversity across bacterial species. A rising analytical approach “pan-proteomics” seeks to evaluate expressed and translated pan-genome [21,22]. Thus, pan-proteomics is presented as an extension of the pan-genome paradigm which permits both qualitative and quantitative proteome comparison, among the species [21,22]. Further, it is well documented that analyses of the pan-proteome not only aids in measuring genetic variations and classification of strain types, but also helps in the identification of vaccine or drug targets and determination of their mode of actions [[21], [22], [23], [24], [25]].

Currently, most of the existing therapies are becoming ineffective with the occurrence and evolution of multidrug and antibiotic resistance in bacterial species [26]. Thus, the need for the current time is to explore the pan-proteome of the bacterial pathogens to identify wide-spectrum therapeutic targets which will be effective against most of the closely linked bacterial species [27,28]. Furthermore, computational biology has emerged as a promising approach for analyzing the genome and proteome sequence data [29,30]. The bioinformatics approaches are more efficient, cost effective, less time consuming, and provides extensive knowledge regarding complete biological systems [[31], [32], [33]]. The current study provided here is focused on in silico identification of probable therapeutic targets from the pan-proteome of eight completely sequenced strains of Ott.