Altered epigenetic landscape as infectious disease diagnostics

1. Challenges in infectious disease diagnostics

Each infection causes pathogen-specific or pathogen class-specific (bacteria/virus/fungi) epigenetic alterations (e.g., DNA methylation, histone modifications, miRNA, mRNA and chromatin remodeling) due to effects on the immune system and other host alterations. Understanding these pathogen induced-human epigenetic changes is a nascent research area, with potential to produce diagnostics with lower limits of detection than direct pathogen testing. Furthermore, host epigenome-based diagnostics have the potential to test for multiple infections simultaneously. As biotechnology continually improves, sequencing based detection systems become faster and easier to use at point of need. Therefore, cataloging the altered human epigenetic landscape due to infection could lead to a shift from pathogen to human epigenome-centric diagnostics.

Molecular diagnostic assays for infectious diseases traditionally query samples for one or a few pathogens at a time. For common illnesses, where a physician can readily diagnose the cause of the disease based on the symptoms, these tests serve mainly as confirmation. However, when a patient has a rarer infectious disease, it can take several iterations of testing to determine the diagnosis, prolonging the time to treatment. Furthermore, molecular diagnostic assays are designed to detect target molecules that are unique to each pathogen, requiring the pathogen to be abundant enough to enable detection by methods such as quantitative polymerase chain reaction (qPCR) or enzyme-linked immunosorbent assay (ELISA); which can limit the doctor's ability to diagnose the disease before symptoms become worse. To address these issues, the diagnostic paradigm can shift to focus on detecting the patient response to infection instead of detecting the pathogen itself. This could enable earlier diagnosis and allow for testing of multiple pathogens simultaneously from one sample. To develop host-based diagnostics, reliable targets are needed to detect and identify the pathogen to enable treatment decisions. Here, the epigenome is discussed as a potential source for infectious disease diagnostic targets.

The epigenome is a series of connected regulatory systems that enable cellular response to environmental changes by initiating or inhibiting gene expression and translation into proteins. The regulatory systems included in the epigenome are DNA methylation, histone modifications, miRNA, mRNA and chromatin remodeling. Methylation of DNA is when a methyl group is added to a cytosine and this can increase or decrease gene expression based on the quantity and location of the methylated bases. DNA methylation regulates gene expression through steric hinderance of transcription factors in promoter regions or through recruitment of repressor proteins [1]. Histone modifications, such as acetylation, methylation, phosphorylation, sumoylation and ubiquitination, cause the protein complexes (histones) around which DNA is wound to move [2]. This shift in histones along the DNA cause regions to become either accessible or inaccessible to gene expression machinery. This dynamic access to DNA is called chromatin accessibility. Transcripts can further be modulated by microRNA which alter ribosome binding, affecting translation into proteins [3]. While miRNAs most commonly induce degradation of the mRNA and repress translation, in some cases miRNA induce translation. All of these regulatory mechanisms converge to enable host response to an environmental change, such as an infection and the epigenetic modifications can last for variable lengths of time, enabling one to determine not only what someone has been exposed to, but an approximate time since exposure. Therefore, by monitoring epigenetic modifications that occur and last for different amounts of time, we can distinguish between acute and chronic infections and utilize the altered epigenomic landscape for infectious disease diagnostics.

2. Infectious diseases alter the human epigenome

Pathogens alter the host physiology to enable ease of access to nutrients, avoid the immune system and transmit to new hosts. Simultaneously, the pathogen triggers the host innate and adaptive immune responses to defend and recover from disease. The host response to a pathogen, as well as the direct alteration by the pathogen, requires several host gene expression changes to take place and therefore the host epigenome is modified upon infection to enable those physiological changes [4,5]. The epigenome regulates the immune response, and several pathogens alter host chromatin accessibility to enable replication. Furthermore, methylation shifts have been shown to be a host response to viral integration into the genome, to reduce viral replication, called epigenetic silencing.

Several pathogens such as human immunodeficiency virus (HIV), Lyme Disease, TB, human papilloma virus (HPV), hepatitis C virus (HCV), uropathogenic Escherichia coli (UPEC) and dysbiosis such as irritable bowel syndrome alter the human epigenome epigenetic states affecting the immune response [6–12]. For example, HIV infection alters the host methylome and has been indicated in accelerated aging and abnormal regulation of immune system regulatory genes [11]. Borrelia burgdorferi, the spirochete that causes Lyme disease, alters the human methylome after infection, with 428 CpGs differentially methylated (256 hypermethylated and 172 were hypomethylated) between infected and uninfected patients [8]. Specifically, three HLA class II alleles were hypermethylated in patients infected with Lyme Disease compared with uninfected individuals, demonstrating the effect of infection on immune system function. Mycobacterium tuberculosis modifies the human macrophage epigenome by secreting its own methyltransferase that trimethylates histone H3K79 and down regulates host methyltransferase activity. This results in an inhibition of apoptosis, an enhancement of necrosis and reduction of host inflammatory and oxidative responses increasing pathogenesis [12]. A respiratory virus, SARS-CoV-2, also affects the epigenome and diagnostics based on methylation were able to differentiate acute infected patients from controls or convalescent patients, as well as to predict long term disease [13].

Some pathogens, oncogenic viruses, not only modify the host epigenome to enable infection, but also influence cell cycle through effects on the epigenome. HPV is an oncogenic virus associated with cervical, anal, penile and throat cancer that affects the human epigenome by two viral E6 and E7 proteins in HPV-associated lesions and cancers [10]. These proteins induce changes in DNA methylation, histone modifications, chromatin remodeling proteins and ncRNAs, resulting in tumorigenesis. Specifically, HPV E7 and E6 affect the activity of human DNMT1. E7 binds DNMT1 and stimulates methyltransferase activity, while E6 upregulates DNMT1 by suppressing p53. The activation of DNTM1 suppresses E-cadherin expression, reducing adhesion between squamous epithelial cells. HPV also affects the activity of several histone readers (BRD4 and PRC), writers (CBP/p300, PCAF, TIP60, EZH2, and SETD7) and erasers (HATs and HDACs 6A/6B) [10,14]. Finally, HPV in cells expressing viral proteins E7 and E6 modifies host miRNA expression globally [9]. Interestingly, miR-203 inhibits HPV replication and is down regulated during infection by E7. Similar to HPV, HCV dysregulates host methylome and histone-modifications. HCV leads to hepatocellular carcinoma through inducting hypermethylation of specific tumor suppressor genes, enabling tumor formation. Again, similar to HPV, HCV dysregulates histone deacetylases causing CDH1 suppression, reducing adhesion between epithelial cells and allowing uncontrolled cellular migration [7]. Thus, there is strong evidence that oncogenic viruses and other pathogens affect the epigenome in ways that can be characterized and differentiated for diagnostic purposes.

3. Innovations in epigenome-based diagnostics

By cataloging and studying the epigenetic changes induced by different pathogens, one could develop infectious disease diagnostic methods which are focused on the altered epigenetic landscape, thus providing a host-based diagnostic for multiple diseases. Currently, methods for querying the epigenome are slow and labor intensive to produce robust data and actionable interpretations of those results. Most methods look at genome wide epigenetic changes, and therefore necessitate the use of high throughput sequencing and intensive computational processing for analysis. However, through development of an infectious disease database of epigenomic signatures, one could enable targeted technologies to reduce the time between sample analysis and diagnosis. Machine learning tools are currently being developed for diagnostic signature identification from the epigenome, including Multiome Accessibility Gene Integration Calling and Looping (MAGICAL) [15] and the Epigenetic Signature Discovery Algorithm (ESDA) [16]. These programs work by sifting through the high-volume data sets to identify and rank epigenetic features that distinguish an infection from an uninfected sample and categorize the epigenetic signatures as belonging to a particular infectious disease based on comparisons to the signatures of other infections. Through these tools, unique epigenetic signatures can be curated into a database from which to build diagnostic assays that distinguish between different infectious diseases. Since most infectious diseases affect the immune system, a blood sample can provide a snapshot of the epigenetic changes occurring in immune cells. Targeted epigenomic assays include methylation or miRNA arrays, methylation sensitive qPCR, miRNA RT-qPCR, or ATAC-qPCR [17]. Furthermore, if global epigenetic signatures are necessary to assess the altered epigenetic landscape for infectious disease diagnosis, nanopore sequencing technology (e.g., oxford nanopore [18]) could be utilized to simultaneously query methylation and chromatin modifications in real time, thereby providing answers to patients within a few hours [19,20].

4. Future perspective

Five to ten years from now, epigenomic signatures will be established for all important infectious diseases, enabling host based diagnostic tests. Healthcare professionals will be able to submit blood samples to a laboratory to determine diagnosis within hours based on the epigenomic signature.

Author contributions

RR Spurbeck is the author of this editorial and is responsible for the intellectual content and manuscript writing. She agrees to take responsibility and be accountable for the contents of the article and to resolve any questions raised about the accuracy or integrity of the published work.

Financial disclosure

This paper was not funded.

Competing interests disclosure

The author, RR Spurbeck, has filed a provisional patent on the ESDA tool mentioned in the editorial under docket 21155USP, the application number is 63/632,390 (filed 4/10/2024).

The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.