Paper: Mitchell A, Tam C, Elli D, Charlton T, Osei-Owusu P, Fazlollahi F, Faull KF, Schneewind O. Glutathionylation of Yersinia pestis LcrV and Its Effects on Plague Pathogenesis. mBio. 2017 May 16;8(3):e00646-17. doi: 10.1128/mBio.00646-17. PMID: 28512097; PMCID: PMC5433101. [link]
Context:
There are three types of plague: bubonic, pneumonic, and septicemic. They’re sometimes thought of as different diseases because they manifest differently in the body, but all three of them are caused by one bacterium: Yersinia pestis. Y. pestis is primarily a rat pathogen, but can be passed from rats to fleas and then from fleas to humans. Bubonic plague can’t be passed from person to person, but bubonic plague can develop into septicemic or pneumonic plague. Pneumonic plague is the most dangerous: it’s in the lungs so it spreads like a coronavirus (i.e., and can be passed from person to person by something as simple as breathing the same air), which is unfortunate because the pneumonic plague is also the most deadly type of plague and has a nearly 100% mortality rate.
There have been three pandemic plague outbreaks throughout history: the first around 541, the second in 1347, and the third in 1894. Each time, it devastated mammals across multiple continents and changed the structure of the society it impacted. The second pandemic is best known as the Black Death, in which around a third of Europe perished. The Black Death of 1347 originated in east Asia and was spread westward over caravan routes, but didn’t pick up steam until rats carrying it made it onto Mediterranean trade ships. Historians and archaeologists argue about if the Black Death was an outbreak of bubonic or pneumonic plague: while the virulence suggests the outbreak was pneumonic, descriptions of the pandemic itself suggest it was bubonic. Because both are caused by the same bacterium, it may be reasonable to assume that it was simply both!
Figure 1. Oops!
Yersinia pestis itself is a deceptively simple bacterium that primarily infects mammals. It has a wide variety of attributes which enhance its invasiveness (i.e., ability to get to a host) and toxicity (i.e., ability to cause problems once it gets into a host) known as “virulence factors.” All of its virulence factors make it more and more dangerous for humans, and one of the most notable of them is the bacterial type III secretion system depicted in Figure 2.
The type III secretion system is like a needle on the surface of a bacterial cell, and allows Y. pestis to inject toxins directly into its host’s cells without any chance of detection by the immune system! It’s made of three broad groups of proteins: first are structural proteins to give it the classic needle shape for maximum ejection ability, second are chaperone proteins to protect and organize unfolded viral proteins within the bacterium, and third are effector proteins to actually interact with the host cell. One key effector protein is LcrV! LcrV is the cap of the type III secretion needle, and it binds to the outer side of the host cell’s membrane.
When something is important to a cell’s basic survival and function, evolution holds onto the genes encoding for it. Scientists have isolated the plague many times, and it’s clear that type III secretion and LcrV are vital to Y. pestis because the genes are relatively constant across the different plague isolates.
Figure 2. The Y. pestis type III secretion needle, from Duncan, Linington, and Auerbuch 2012.
The type III secretion system is not unique to Y. pestis, but Y. pestis uses a specialized version of it. This makes Y. pestis more dangerous to mammals, because its uniqueness means it’s less likely to be recognized by a mammal’s immune system. This 2017 paper is one of many studies characterizing the differences between Y. pestis and other bacteria, and does so by studying LcrV on Y. pestis.
LcrV:
In this paper, Mitchell et al. argue that LcrV has been glutathionylated at a cysteine, which is the 273rd amino acid in the protein (C273). Glutathione is a small reducing agent made from three amino acids: glycine, glutamate, and cysteine. Since it includes cysteine, glutathione is a thiol, which means it has an -SH group on it. This is valuable because sulfur can form some really strong bonds, so glutathione can attach to things pretty securely. To place the strength of these bonds in context, disulfide bonds (i.e., bonds between one sulfur and another sulfur) like these are central to the protein keratin, which makes up our hair, nails, and much tougher things like rhino horns. One reason that the horns in particular are so tough is a high prevalence of disulfide bonds formed by cysteine residues (in this context, “residue” refers to a single, specific amino acid) in the protein. There are a couple of different ways that glutathione can serve the protein that it is attached to. Firstly, the glutathione can protect the cysteine it’s attached to by keeping it from reacting with something else. It is like giving kids waiting for the dentist something to color with, even though coloring won’t necessarily help their teeth. In LcrV specifically, it is possible that C273 has to take part in a different, more important reaction later on, so protecting the cysteine side chain chain from oxidation is important.
Figure 3: The chemical structures of the small peptide glutathione and amino acid side chains for cysteine, alanine, and serine.
Article Summary:
Several important experiments in this paper depend on the mutation of this cysteine, as scientists tend to mutate proteins to examine the importance of individual amino acids to the overall function of the protein. In this paper, the researchers mutated the cysteine to an alanine (C273A) and to a serine (C273S). Alanine has only a methyl (-CH3) group on its side chain (no sulfur!) and therefore is not able to form disulfide bonds. Serine is more similar to cysteine, only with an -OH alcohol group instead of an -SH thiol group at the end. Unfortunately, oxygen is still not able to make bonds as strong as the disulfide ones!
The researchers propose that the glutathionylation of C273 in LcrV is important to plague infection. They began by weighing small sections of this protein to test if C273 is glutathionylated, and were able to determine that the weight of a small section containing C273 had extra mass not accounted for by the amino acids in the chain. This section differed from the weight of just the amino acids by roughly the weight of one glutathione. Perhaps, more interestingly, the authors were able to show that when a mutation was introduced at C273 to change it to alanine (C273A), it took longer for the rodents to be killed by the plague.When this mutation was made, the residue could no longer form disulfide bonds and therefore could no longer be glutathionylated. This finding suggests that this residue, and perhaps even glutathionylation of this residue, is important to plague pathogenesis.
Once they had determined that C273 is glutathionylated, the scientists had to prepare multiple “strains” (sub-types) of the plague and select the types of rodents to test. They used a strain of the plague isolated from a fatal human case, which is denoted by “wild type” (WT). They modified this strain by mutating C273 in LcrV to an alanine, which is denoted by “C273A” strain. The rodents used included lab mice, which are highly susceptible to infection by Y. pestis, and rats, which are moderately resistant. Including both species allowed the scientists to see how the infection of organisms susceptible to infection compared to those less susceptible. It also allowed the scientists to easily compare the time to death and the disease progression in different organisms. The mice showed the time to death comparison well, while the rats allowed more time to investigate the stages of the spread of the bacterium in the body. Interestingly, in this paper, both types of rodents survived longer when given the strain with the C273A mutation.
Figure 4: Glutathionylation of LcrV enhances bubonic plague pathogenesis. A) shows survival of BALB/c lab mice who were injected with a standard amount of Y. pestis while B) shows survival of Norway lab rats injected with a standard amount of Y. pestis. In both studies, two different strains of Y. pestis were used. One was isolated from a fatal human case of the plague (WT) while the other (C273A) was identical to WT, only with a cysteine to alanine mutation at C273 in LcrV. Adapted from Figure 3 in Mitchell et al (2017).
Mitchell et al. were able to connect the importance of C273 to broader observations made of Y. pestis in nature. There is a specific variant of Y. pestis called Microtus, which does not cause the plague in humans. In some Microtus isolates, there is a C273S mutation, where serine is substituted for cysteine. This is interesting because, as previously mentioned, serine has an oxygen instead of sulfur and therefore can not be glutathionylated (Figure 3). An additional study further emphasized the importance of this part of LcrV. C273 is within a broader region of LcrV which is highly variable in nonepidemic strains of Y. pestis. This suggests that in bacteria which don’t cause highly transmissible plague disease, this region changes more. The authors of this paper seem to hint that this suggests that in order for the plague to be infectious, this region cannot vary. (The authors of this blog post think this could be a bit of a reach, but is an interesting idea to look into!)
Conclusions, Implications, and Future Studies:
Mitchell et al. concluded that the presence of a single glutathione group attached to a cysteine on the LcrV protein increases the deadliness of the bubonic plague in rodents. Glutathionylation increases the number of virulent proteins that are injected into cells by Y. pestis and promotes Y. pestis growth in mammalian blood, thereby increasing its virulence and decreasing the time from infection to death.
There are still plenty of experiments that remain to be done on LcrV glutathionylation! It is likely that LcrV accesses one of its host’s ribosomal proteins using the type III mechanism, but the details of that access are still unknown! Given the importance of type III secretion to Y. pestis virulence, future research surrounding this is vital. Additionally, more work can be done to look at why glutathionylation of C273 on LcrV assists in Y. pestis pathogenesis. The authors predict that this might be due to a reduced immune response to C273A. The bacterium depends on the immune system to spread it around the host. Therefore, when the bacterium isn’t able to flag down a ride on a macrophage (or other immune system cell), there is less spread of the bacteria throughout the body. This hypothesis could be examined by looking more closely at methods of plague distribution in the body and the presence of both WT and C273A Y. pestis in immune cells. Finally, these experiments were only done in Y. pestis, so it’s unknown if LcrV is glutathionylated in other pathogenic bacteria. It may be universal, or it may be unique and one of the factors that makes the plague so dangerous.
About the Authors:
Katherine Dailey (she/her; ‘22) is a biochemistry major and environmental studies minor. At Mount Holyoke, she is a member of the Berry Lab and the rowing team. This spring, she will be dedicating herself to microbiology full time as she heads off to study bacterial pathogenesis in a lab at Harvard Medical School.
Jaya Nagarajan-Swenson (they/them, ‘22) is an astronomy and biology double major. At Mount Holyoke, Jaya is a coxswain on the rowing team (with Katherine!), works in the Fimbel Maker & Innovation Lab, and is studying dark matter. This fall, they will be putting the bacteria down (☹) and starting a PhD in extragalactic astronomy at the University of Virginia.
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