While scientists using ancient DNA analysis are learning how Yersinia pestis developed over time into the causative agent of three worldwide pandemics, there is still much to learn about the early hours and days of an organism infected with the plague. Y. pestis still infects humans so any insight into disease progression is useful for determining treatment timing and even developing novel treatments to supplement or replace antibiotics. A 2012 study observed how Y. pestis injected into mice spread throughout the body using bioluminescent imaging to track the infection. More recent research reported in PLOS ONE used a more real-world route of infection by introducing an aerosolized Y. pestis to a nonhuman primate model and tracking the transcripts altered during the first 42 hours of infection.
To get a baseline of gene expression, blood was collected from African green monkeys, the nonhuman primates studied, 24 hours prior to infection with Y. pestis. The experiment mimicked a pneumonic plague infection route by nebulizing Y. pestis in saline and having anesthetized fasting monkeys breathe in the measured infectious dose. Three animals were exposed to saline as a control for the aerosolized infection. Blood was drawn at 45 minutes, 6 hours, 9 hours, 12 hours, 18 hours, 24 hours, 32 hours and 42 hours post-exposure with three or four primates euthanized at each of time points after 45 minutes. Two infected animals were allowed to progress to death at 78 hours, marking the end point for the infectious dose administered to the primates.
Blood, urine and nine different tissues from the infected primates were cultured to determine bacterial load. Colonies were detected from blood at 9 hours with all animals growing colonies from blood collected at 32 hours or more. Liver and lung tissue had detectable colonies at 6 hours.
Total RNA was isolated from blood samples, the transcripts pooled for each time point and the cDNA labeled and used to probe a custom rhesus monkey oligonucleotide microarray. Blood samples taken 24 hours prior to Y. pestis exposure were used as the transcriptional baseline. The microarray transcripts that were altered fell into two categories: Early infection and late infection. That is, the expression profile for 45 minutes–18 hours (early infection) post-Y. pestis exposure altered one set of transcripts, while 24–42 hours (late infection) after Y. pestis infection affected a different collection of transcripts. These transcriptional changes were detected even before bacteria could be cultured from blood, indicating Y. pestis quickly affects its host after infection. Not surprisingly, genes associated with natural killer (NK) cell signaling and macrophage phagocytosis were inhibited, several cytokines were gradually suppressed and apoptosis signaling was activated during early infection. These changes would likely curtail the innate immunity, allowing Y. pestis infection to proceed unchecked. In late infection, DNA repair was activated, mTOR, AMPK and p53 signaling networks were suppressed and elevated expression of chemokines and cytokines indicated sepsis, a common consequence in the late stages of Y. pestis infection.
Hammamieh et al. also compared the transcripts between uninfected and infected blood samples, the latter by pooling all the blood sample time points, and identified several signaling networks affected. These included apoptosis, microtubule stabilization, inflammatory leukocytes recruitment, protein ubiquitination, G2/M DNA damage checkpoint and p53 pathways. The widespread activation or suppression of such a diverse set of genes shows how Y. pestis can profoundly affect an organism, disrupting cellular signaling and leading to death.
qPCR was used to confirm the microarray results, selecting a subset of genes involved with ubiquitination, cytokine signaling and apoptosis among others. The ten genes amplified in qPCR showed similar gene expression changes for most of the time points, supporting the results demonstrated in the microarray.
Blood chemistry testing of infected samples compared to the 24-hour preexposure baseline showed rapid changes in the panel of analytes. Hammamieh et al. divided the samples into early and late infection, finding not only changes compared to baseline, but changes over the course of 45 minutes–42 hours postinfection. While blood urea nitrogen (BUN) and creatine kinase (CK) were elevated for both infection times, lactate dehydrogenase (LDH) and alkaline phosphatase (ALKP) increased during early infection, but became baseline in late infection. These indicators parallel the gene expression changes, demonstrating the consequences of the activation and repression of the signaling pathways during Y. pestis infection.
Using a pneumonic plague infection route in nonhuman primates, Hammamieh et al. showed how Y. pestis alters gene expression even 45 minutes after exposure to the bacteria. By inhibiting the innate immunity of NK cells, activating the inflammatory immune response and altering pathways involved in apoptosis, DNA repair and cell structure, Y. pestis infection wreaks havoc from the early exposure though the later stages of disease. This is just preliminary work, but this transcriptional analysis helps scientists begin to understand network of genes altered by infection and devise strategies for blocking infection before it becomes systemic.
Hammamieh, R. et al. (2016) Temporal progression of pneumonic plague in blood of nonhuman primate: A transcriptomic analysis. PLOS ONE 11, e0151788. doi: 10.1371/journal.pone.0151788