Caitlyn Coleman

Introduction

Archaeological research has had a considerable impact on modern discoveries. Studies on skeletal remains and mummified samples led to improvements in the research process for modern infections. Ancient DNA research has its challenges with controlling contamination and the fragmentation of the samples, but improvements in methodology such as Uracil DNA glycosylase (UDG) treatments [Llamas et al, 2016] have mitigated damage in some regard. However, it is necessary to consider more than  just DNA, but a holistic approach that includes proteomics, metabolomics, and genomics. The objective of this literature review is to synthesize the advances in these three studies with an emphasis on infectious diseases. There is specific attention paid to the bacterium Yersinia pestis,  and the Black Death.

Proteomics in Archaeology

Applications within Mummification 

 Protein networks within ancient DNA contain vital information to better understand infectious diseases. The identification of various proteins and the discovery of new putative proteins furthers archeological research.  The presence of certain proteins can indicate an immune response or point to inflammation, which demonstrates possible infection [Jones et al, 2016]. The most abundant category in a study using  ancient Egyptian mummies was anatomical structure development, specifically collagens and keratins [Jones et al, 2016]. Another study on a Korean mummy used genomic analysis to pinpoint four open reading frames (ORFs) that aligned with the hepatitis B virus [Bar-Gal et al, 2012] . The strain of hepatitis B virus that was mummy-derived was compared to modern DNA to establish how long the virus had been in East Asia and how it had evolved over thousands of years [Bar-Gal et al, 2012]. Through bioinformatics analysis, specific primer sequences found in Inca mummy samples were matched to genomes in the phylum Actinobacteria [Corthals et al, 2012]. Gel electrophoresis confirmed the results  with two lanes producing positive results that matched Mycobacterium tuberculosis [Corthals et, 2012]. These studies on mummies from different countries has increased the knowledge of pathogen evolution and the spread of infectious diseases in both past and present times.

Fifteen immune response proteins, three stress-response proteins, and twenty-six metabolic proteins were found through proteomics analysis of muscle tissue biopsies of an adult mummy [Jones et al, 2016].  These metabolic proteins included enzymes such as Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Amylase which both connect to inflammation [Jones et al, 2016]. Remains alone have not been able to reveal the presence of disease, but in combination with aDNA analysis, stronger results have been produced.

Microbiological Application within the dental calculus

The dental calculus provides insights to the microbiome of ancient civilizations. It allows researchers to document disease throughout history. Specifically, dental plaque holds hundreds of types of bacteria [Preus et al, 2011]. While it is noted that there is stability in the oral microbiome, it is one of the most accessible areas for pathogens to enter the body [Preus et al, 2011]. Germs can easily be transferred from hard surfaces to people’s hands, but from here, they must find a way inside. One way is through improper handwashing, or its absence altogether, and then touching one’s face or food. Deadly infections can be transmitted this way, and even in death, the killer microorganism can be found in the victim’s saliva [Preus et al, 2011]. It was found that the protein taxonomy has variability, and certain categories are expected to show up more often [Mackie et al, 2017]. While pathogenic bacteria were identified, that was only one of three sources of bacterial proteins. [Mackie et al, 2017]. Other sources included opportunistic and commensal species of bacteria. [Mackie et al, 2017]. New studies have the potential to disclose mechanisms involved in the evolution of microbiome bacteria [Weyrich et al, 2015]. The dental calculus preserves the pathogen of that time.

Dietary Application within the dental calculus

Dental calculus also provides information on the diet of the specimen. It can narrow down specific food groups that were consumed more often than others. Fragments of the food consumed are preserved in the dental calculus deposit along with biomolecules and diagnostic proteins [Hendy et al, 2018]. Many diseases are transmitted from animals to humans, so it is important to note what species were a main food source during the time period of study. The microfossils within dental calculus hold details about the evolution of various plants and animals that made up the dietary behavior of various cultures [Weyrich et al, 20 15]. The dental calculus is a record of the dietary habits of the people it is sampled from.

Application using Yersinia pestis and  Yersinia pseudotuberculosis

In Yersinia pestis, tracking the activity of the urease enzyme led to a discovery that explained how the plague evolved over time. This enzyme played a key role in the transmission of the bacteria throughout Europe by fleas [Chouikha and Hinnebusch, 2014]. Urease is toxic to fleas, and its deletion increased the potential for transmission, hence the mutation was favored in Yersinia pestis [Chouikha and Hinnebusch, 2014]. In a past study the results proved that the presence of functional urease is a requirement if the bacteria Yersinia pseudotuberculosis was to have any toxicity in fleas [Chouikha and Hinnebusch, 2014]. Urease enzymes have further explained the evolution of the Black Death.

Metabolomics in Archaeology

Infectious Disease Application

Metabolic processes play an important role in the study of infectious diseases. They are interrupted by the infection, and that disruption impacts the end products or metabolites [Ghost et al, 2018]. The sensitivity of these pathways to changes in the environment, or increased stress make the infection progress easily visible [Ghost et al, 2018]. In the case of malaria, the effect on the host’s ability to utilize glucose starts a chain reaction that lowers the production of creatine, increases the levels of creatine phosphokinase (CPK), and trickles down to a decrease in phosphocreatine synthesis [Ghost et al, 2018]. In addition, metabolites from Brown propolis used in a biofilm were successful in decreasing the amount of Staphylococcus aureus and  T. vaginalis [Dembogurski et al, 2018]. Phenylpropanoic acids and flavonoids are two significant compounds that caused the decrease in bacterial activity [Dembogurski et al, 2018]. Metabolites are a way to follow infectious disease through the biological systems of their host.

Lipidomics Subset

Lipidomics concentrates on how lipids interact with the metabolites of metabolomics. 

Two lipids that hold great significance in the study of infectious diseases are phosphatidylcholine (PC) and phosphatidylethanolamine (PE) [Wang et al, 2019]. Phospholipids are also important to monitor as any fluctuation in their levels detect changes in an organisms’ overall functions [Qin et al, 2016]. Analysis of lipids often involves either radioactive or fluorescent labeling to keep track of the sample [Lam and Shui, 2013]. It also requires mass spectrometry and chromatography to have a full lipidomic approach [Lam and Shui, 2013]. Lipid interaction is another method of tracing the spread of an infection through the host organism.

Genomics in Archaeology

General Genomics

Genomics is the study of an organism’s genes in their entirety: the genome.  There are complications that arise when dealing with ancient DNA genomes that make it difficult to show the diversity in various pathogens responsible for past outbreaks [Andam et al, 2016]. Uracil selection (otherwise known as U selection) is a new method that has shown promise in decreasing contamination in ancient genome research [Gansauge and Meyer, 2014].

However modern outbreaks such as the Ebola and Cholera outbreaks have benefitted from genome sequencing [Andam et al, 2016].  Genes hold much more than genetic material, they have the potential to improve infectious disease research of both ancient and modern pathogens.

Applications  using Yersinia pestis

Genomic analysis of Yersinia pestis allows researchers to better understand how it spread so rapidly across Europe. Three historical genomes studied contained specific single-nucleotide polymorphisms (SNPs), within the pabA and  NCa genes. [Spyrou et al, 2016]. These genomes ended up on the same phylogenetic branch, even though they originated from Barcelona, Bolgar City, and Ellwangen [Spyrou et al, 2016]. It was found that when comparing Yersinia pestis to Yersinia pseudotuberculosis, isolates of pestoides and microtus were more closely related to Yersinia pestis. [Atchman et al, 2004]. The genome of Yersinia pestis provided more insight to how it caused  multiple pandemics.

Current Methodologies Flowchart

Significant Research Questions

After extensive review it was clear there is commonality among the research questions being pursued. These common questions include:

1. How can contamination be minimized in archaeological research?
2. What is the most effective combination of treatments to most accurately study ancient DNA?
3. What can archaeological research reveal to improve modern studies?

CONCLUSION

In this review, proteomics, genomics, and metabolomics, were outlined as they relate to infectious diseases, and specifically Yersinia pestis. The current methodologies and significant research questions were compiled. None of these disciplines are sufficient alone, but a combination of the three yields more comprehensive research. Proteomics and metabolomics allow a closer look at the impact of infectious diseases on biological systems. Genomics and genome sequences track the spread of these diseases and compare different strains. 

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