The most infamous plague in history is the Black Death, otherwise known as the bubonic plague. Despite its historical fame, the plague is still present today. The CDC reported about 1000 human plague cases between 1900 and 2010 in the USA for people of all age groups; averaging about 7 deaths per year. One of the deadliest diseases ever, the plague is caused by a small, unassuming bacteria, Yersinia pestis, through fleas and rodents. Y. pestis is a rod-shaped, nonmotile, gram-negative bacterium (Figure 1). The cell wall structure of a gram-negative bacteria is a thin peptidoglycan wall sandwiched between an outer and inner membrane.
Figure 1. Gram stain of Y. pestis
Y. pestis transmission happens when a flea is infected through feeding. The infected fleas with the bacteria go on to infect humans and rodents–rodents will also infect humans. A part of Dewitte et al’s study published in PLOS Pathogens 2020 assessed how the consumption of meals with Y. pestis affects the accumulation of a brownish mass of the bacteria in the proventriculus (glandular stomach where digestion begins). What they found suggested that the flea infection by Y. pestis produced a bacteria mass in the proventriculus causing a blockage as described in Figure 2. The bacteria mass in the proventriculus in the soft form gets dislodged into the midgut (where digestion occurs) through feeding by the flea. With time, there was an increase in free-floating masses of bacteria in the shape of the proventriculus cast in the midgut. While dislodging happens, a new mass of Y. pestis infection is cast in the proventriculus and the dislodging cycle repeats until the mass of bacteria at the proventriculus is strong enough to resist the flow of any materials, particularly blood. This leads to the blockage of the proventriculus of the flea. Blood was reported as the main material that dislodged the mass, but it was also hypothesized that fleas had the ability to dislodge a small amount of growing cast themselves. As to how fleas dislodged the case, it was not reported. An analysis that was reported stated that the more meals a flea had, the more mass production increased, and the size of the mass anchored to the proventriculus decreased.
Figure 2. A physical model of the processes leading to flea blockage by Y. pestis. (Adapted from Dewitte et al)
After discovering the mass formation cycle, they started to look into the specific genes involved in forming a strong mass blockage in the proventriculus of the fleas. They soon discovered that only specific genes of Y. pestis were able to form the mass in the proventriculus. This was discovered by feeding fleas with blood not containing bacteria or blood containing other bacteria, some of which shared ancestry with the Y. pestis. They discovered no blockage in the fleas or infections for both blood samples. Based on these results, they were able to determine a gene called ymt–produced by the flea–was involved in trapping the bacterial mass blockage in the proventriculus.
The next agenda was to find the mechanism Y. pestis used in the progression of the fleas’ blockage. Based on transcriptomic analysis reporting genes in Y. pestis, mutations at varying sites–including neighboring genes–were made to scale down to genes that were required for biofilm formation which leads to the blockage in the proventriculus. Biofilm formation happens when bacteria come together to create a multicellular organism with the goal of protecting them against antimicrobial and immune responses. Out of nine genes identified, rpiA and rpiA with rpiA2, were significantly linked with blockage formation after biofilm formation (Fig 3).
Figure 3. Biofilm formation and blockage rate in mutant fleas (Adapted from Dewitte et al. 2020)
They hypothesized that rpiA is required for resistance against the flea’s immune system. Through experimentation, data suggested that optimal colonization of the bactericidal mass produced in the proventriculus requires rpiA to confer resistance to harmful compounds such as antimicrobial peptides. Antimicrobial peptides would otherwise dislodge and destroy the foreign bacteria. They also found that rpiA expression levels were involved in forming the mature biofilm needed to block the flea’s proventriculus. The results correlated with blockage rates and increased the spread of the plague. The ΔrpiA and ΔrpiA-rpiA2 mutant fleas had lower levels of blockage formation compared to the WT fleas (Fig 3). Additionally, ΔrpiA mutant fleas had an easier time clearing the blockage from their proventriculus than WT fleas, supporting the hypothesis that rpiA helps anchor and solidify the mass to the resistant movement of the proventriculus and blood flow.
As some blockage formation was possible with the ΔrpiA mutant fleas, rpiA is not necessary for the formation of the blockage. However, as demonstrated in Figure 4, rpiA aids with consolidating the blockage. A secondary rpiA, rpiA2, gene was found in the experiment from Figure 3. rpiA2 accounts for about 15% of the blockages while rpiA accounts for 85%. Further experimentation was performed, looking at the mass formation in the proventriculus in WT, ΔrpiA, and ΔrpiA rpiA2 fleas (Figure 5).
Figure 5. The proventriculus casts contain WT, ΔrpiA, and ΔrpiA-rpiA2 Y. pestis. (Adapted from Dewitte et al. 2020)
With a combination of fluorescent and bright-field microscopy (and the power of Photoshop), Dewitte et al were able to show the formation of blockages in the fleas from three different genotypes: WT, ΔrpiA, and ΔrpiA rpiA2. The proventriculus is autofluorescent in green (it’s automatically green under fluorescent microscopy, no Photoshop magic needed). The bacteria are highlighted in blue, thanks to Photoshop. The denser the blue and the brighter the color, the more consolidated the bacterial masses. The WT flea, which expresses the genes rpiA and rpiA2, shows the most consolidated mass formation. The ΔrpiA fleas show some mass formation, but there is hardly any blockage of the proventriculus, and the bacterial mass seen is wispy. The ΔrpiA rpiA2 fleas have the least amount of blockage of all the genotypes, demonstrating the necessity of rpiA, or at least rpiA2, in the blockage of the proventriculus.
The mass floating in the gut of the flea are bacterial masses that detached from the proventriculus. The same pattern can be observed with the floating masses as with the proventriculus blockage, with WT having the most consolidated floating mass and the ΔrpiA rpiA2 fleas with the least consolidated.
Knowing that rpiA and rpiA2 are so essential in the formation of the block in the proventriculus provides scientists with a new target for the prevention of Y. pestis infections and plague outbreaks. Physical and genetic differences within fleas are another target of experimentation to further confirm the results of Dewitte et al’s study. To stop plague outbreaks, the ability for the bacteria to be transferred from animals to humans must be halted. Fleas are one of the main sources of infection of the plague in both rodents and humans. However, in order for Y. pestis to get transferred to a new host, it needs to force the flea to regurgitate the bacteria. For regurgitation, the bacteria need to block the proventriculus. If scientists are able to develop a way to prevent the blockage of the flea proventriculus or reduce the amount of blockage, it would greatly hinder the spread of the bacteria that cause plague. But confirmation of these results and further study is necessary before such treatments are discovered.
Ama Boamah '23 is a Chemistry major and Biology minor on a Pre-pharmacy track at Smith College. When Ama is not in the research lab of Prof. Buck working on her honors thesis on Protein Polymer Conjugates, she's crocheting, hanging out with friends, watching movies, or making drinks. Ama is from New York City and will pursue her Pharmacy Degree after graduating this fall.
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