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Introduction
Yersinia pestis, the causative agent of plague, has been historically accountable for greater than 200 million deaths throughout three pandemics. Zoonotic maintenance of plague occurs through the ability of it propagation and circulation amongst rodent reservoir hosts and flea vectors. Upon consumption of an infected mammalian blood meal by a naïve flea, Y. pestis proliferates in the flea midgut. Y. pestis forms a biofilm in the flea proventriculus which prevents the passage of blood during subsequent feeding attempts. Moreover, the biofilm enhances the regurgitation of bacteria into the dermis of the mammalian host, thereby promoting the natural transmission of plague.
Y. pestis strains can be assigned to one of four biovars: Antiqua, mediaevalis, Orientalis, and Microtus. With biovar, Orientalis being the most recent evolutionary divergent variant believed to be the cause of the 3rd plague pandemic. It is the only variant known to have lost its capacity to ferment glycerol.
Fig1: Metabolisms in Different biovars
Prior transcriptomic analyses indicate that the components of the aerobic glycerol metabolic pathways are induced during multiple aspects of the Y. pestis infectious cycle. Aerobic glycerol metabolism is simulated during temperature shifts representing the transition from the flea vector (26°C) to the human host (37°C) and amid survival in the macrophage intraphagosomal environment.
Cell Structure
Yersinia pestis is a rod-shaped gram-negative bacterium that can also have a spherical shape. It falls under the Coccobacillus category of bacteria, with no spores. It is also covered by a slime envelope that is heat labile. When the bacteria are present in the host, they are nonmotile (incapable of self-propelled movement), but when isolated they are motile. It is a Facultative Anaerobic Organism and was first discovered by Alexander Yersin, a Swiss/French physician and bacteriologist from the Pasteur Institute. Similar to other Yersinia species, it tests negative for urease, lactose fermentation, and indole. The closest relative is the gastrointestinal pathogen Yersinia pseudotuberculosis, and more distantly Yersinia enterocolitica.
Pathology
Yersinia pestis interacts mainly with rodents such as rats and fleas. Through these carriers, Yersinia pestis is able to invade human cells and create diseases. Yersinia pestis are not rich in nutrients and can grow at temperatures ranging from about 26 Celsius to 37 Celsius.
Y. pestis causes diseases through the bite of an infected rat or flea, but can also be transmitted by air. Fleas can become infected by taking the blood of other infected animals. Y. pestis grows in the midgut and eventually blocks the proventriculus, starving the flea for blood. The insects attempt to feed more often but end up giving back infected blood into the wound of the bite.
The major defense against Y pestis infection is the development of specific anti-envelope (F1) antibodies, which serve as opsonins for the virulent organisms, allowing their rapid phagocytosis and destruction while still within the initial infectious locus. The immune mechanism against this disease is extremely complex and involves a combination of humoral and cellular factors. The host is immune to secondary virulent attack, the inoculum being eliminated as though the organisms were completely avirulent.
Metabolisms
(*Note: Yersinia pestis is a facultative Anaerobic organism which means that it gets ATP via Aerobic respiration if oxygen is present and if oxygen is absent it can switch to fermentation for its ATP requirements. *)
1. Glycolysis
Early studies of metabolism in YP focused on pathways of carbohydrate consumption. It was indicated that resting cells of YP utilize glucose primarily via the Embden-Meyerhof-Parnus pathway and that alternate pathways such as pentose phosphate pathway (PPP), do not contribute to this process. YP was observed to have the enzymatic capacity to catalyze all the constituent reactions of the glycolytic pathway.
In YP the traditionally recognized terminal step of this pathway, as catalyzed by the enzyme pyruvate kinase, might not be as extensively used for conversion of phosphoenolpyruvate (PEP) into intermediary metabolites of the citric acid cycle. Instead, YP tends to use the enzyme phosphoenolpyruvate carboxylase (Ppc) to carboxylate PEP into oxaloacetate. It was indicated that balance of oxaloacetate in YP 24 Metabolism of Yersinia pestis is a critical factor in cellular growth. Any metabolic perturbation that results in accumulation of oxaloacetate tends to encourage cellular growth while depletion of oxaloacetate tends to have the opposite effect.
This deduction is strongly supported by the observation that presence of CO2 or bicarbonate, stimulates cellular growth. In YP the reliance on Ppc could also alleviate some of the harmful consequences of aspartase deficiency. This is because absence of ASPA means that any process that would convert oxaloacetate into aspartate would be diverting metabolic carbons into the production of a dead-end product.
2. Pentose phosphate pathway
Initially, it was reported that all the constituent enzymes of PPP are present and active in YP and that during the growth phase this pathway is used to provide the pentose phosphates that are necessary for production of biomass. These conclusions were based on experiments where using 1- 14C-glucose with an avirulent strain of bacteria, it was discovered that the amount of 14C labeled CO2 released by growing YP was nearly 4 times greater than that liberated by resting cells. The presence of all enzymes of PPP was surmised since conversion of carbon 1 of glucose to CO2 results from oxidative decarboxylation by the pentose phosphate enzyme, phosphogluconate dehydrogenase.
However, later studies with the virulent Alexander stain showed that although cell-free extracts of YP include a number of different enzymes of the pentose phosphate pathway, the activity of glucose-6-phosphate dehydrogenase could not be detected. This observation has been confirmed in over 50 different YP strains spanning diverse geographic locations and all virulent biovars Contemporary sequence analyses of the YP genome have found a mutation in the gene encoding this enzyme (a proline substitution in amino acid 161), which supports the latter observation.
3. Anaerobic Metabolism
Under ordinary usual circumstances, YP grows in an oxygen-rich environment. These bacteria are facultative anaerobes and can also grow via fermentation. At 26°C, expressions of a number of genes associated with anaerobic energy production (such as fumarate and nitrate reductases) are upregulated. Although this could indicate that in the flea gut environment, oxygen is sparse and that YPs metabolism shifts to anaerobic respiration upon transmission into this medium, absence of concomitant increases in expression of regulatory proteins response to anaerobic conditions have led to postulation that the increase in expression of above mentioned metabolic enzymes is primarily due increased cellular growth at 26°C (in comparison to LCR conditions).
Oxidative metabolism of glucose has been shown to produce very small quantities of organic acids, anaerobic carbon metabolism in contrast is highly inefficient. In the absence of oxygen, the metabolism of glucose will result in production of a number of organic acids, such as acetate, lactate, and formate. Overall, while oxidative metabolism of glucose results in nearly 60 percent assimilation of carbons into the cellular biomass, only 40 percent of the carbons are assimilated via anaerobic metabolism. As can be expected, resting cells in anaerobic environments cease to maintain a viable store of enzymes associated with TCA cycle and associated processes for oxidative mode of energy production.
4. Citric Acid Cycle & Oxidative metabolism
YP has a functioning TCA cycle. Observations indicate that given YPs nutrient-rich environment, it frequently utilizes oxidative metabolism for its energy needs, for example, in vitro aeration of YPs medium increases its growth rate. Growing YP rapidly oxidizes glucose and pyruvate while oxidative metabolism of acetate, succinate, fumarate, and malate proceeds at a lower rate. Finally, microarray gene expression data have shown that induction of cellular growth following the transition of cells from LCR to calcium-rich and 26°C conditions are accompanied with increases in expression of genes associated with oxidative modes of energy metabolism.
Even resting cells of YP, if grown aerobically, have a high rate of endogenous respiration (15-30% of respiration in presence of nutrients). This process proceeds independent of exogenous metabolism and thus the rate of O2 uptake and CO2 release is not a direct measure of the metabolism of nutrients from the media.
5. Glyoxylate Shunt
Although no enzymatic deficiencies have been reported, it was experimentally indicated that the YP citric acid metabolism does not proceed via the traditional reactions associated with the TCA cycle. Instead, the bacteria utilize the glyoxylate shunt as an alternate means. The glyoxylate shunt of TCA cycle is often used as an anabolic pathway for conversion of two-carbon compounds (such as acetate) into glucose via the metabolism of acetyl-CoA. Results support the notion that YP normally uses this pathway for its metabolic needs. First, it has been observed that YP can fully oxidize acetate with negligible production of ±-ketoglutarate, and secondly, YP extracts have a limited capacity to convert ±-ketoglutarate into succinate. Computational simulations of cellular metabolism also indicate that in rich media, there are a number of viable alternate pathways for the metabolism of oxaloacetate.
Enzymes essential for the glyoxylate bypass pathway are isocitrate lyase and malate synthase. Unlike E. coli and other Yersiniae, YP maintains significant levels of both of these enzymes during growth on hexose and pentose molecules. In YP, two forms of isocitrate lyase have been detected. One form is active during growth on acetate and absents when alternate carbon sources are utilized. The other form of the enzyme is not constitutive but can be detected while growing on a variety of different carbon sources. Therefore, it has been postulated that this form might have a significant role that is not associated with the enzymes traditional anaplerotic function
Finally, microarray analyses of gene expression associated with the glyoxylate shunt show that three of these genes (aceK, icl, and mas) are upregulated upon in 37°C LCR conditions. *(This could indicate that metabolism of two-carbon metabolites is more pronounced in the mammalian host; however, this hypothesis has not been verified) *. Thus, it can be argued that, although YP possesses the enzymatic means to proceed via traditional TCA cycle processes, it normally uses the glyoxylate shunt and bypasses some of the formers reactions. The reason for deviation is not known. However, it has been hypothesized that given the glyoxylate shunts role as a means to convert two-carbon organic compounds into TCA cycle intermediates, the metabolic change could serve as a mechanism to replenish oxaloacetate that is withdrawn from cellular metabolism via its conversion into ASP. This further supports the assertion that absence of ASPA (which would have recycled ASP back into a TCA cycle intermediate) is a central factor in how the overall metabolism of the bacterium operates.
6. Amino Acid
i. Missing amino acid biosynthesis pathways
The principal pathway for incorporation of NH4 + into cellular metabolome and biomass occurs through the action of the enzyme glutamate dehydrogenase.
Following this initial step, the resultant glutamate (GLU) can be converted into other amino acids and intermediates of the TCA cycle through a number of different transamination reactions. Wild types ceased to grow in media which have low levels of ammonium salts as the sole source of nitrogen. It has been proposed that this mesotrophic deficit can be attributed to mutationally induced inactivation of glutamine synthase.
The loss of this metabolic capability is yet another indication that YPs metabolism has begun to adapt to nutrient-rich environments. Furthermore, it indicates that the bacterium prefers to import a large portion of its amino acid nutritional needs from the surrounding medium. In line with this metabolic program, YP has lost the capacity to produce a number of amino acids and under normal conditions requires their import for cellular growth. YPS does not have an obligatory requirement for import of these amino acids and thus it can be deduced that YPs transition to living in the nutritionally rich fluids has resulted in loss of capabilities which are essential for survival of its free-living progenitor.
ii. Use of amino acids as sources of carbon and nitrogen
Although only the import of some of the important amino acids is obligatory for cellular growth *(glycine (GLY) or threonine (THR), methionine (Met), phenylalanine (PHE), cysteine (CYS), isoleucine (ILE) and valine (Val)) *, a number of other amino acids can be metabolized by YP as carbon and nitrogen sources. For example, YP can rapidly catabolize SER via production of pyruvate and acetate intermediaries. Some studies have identified arginine (ARG) as one of the amino acids that cannot be catabolized by both YP and YPS. However, this metabolism is critical for YPs transitions between flea gut and host environments. Microarray gene expression studies show that at 37°C, some of the genes involved in ARG biosynthesis (argABC) and its interconversion to GLU (astCADBE) are upregulated. The latter process releases CO2 and ammonia as by-products which can be interpreted as an added cellular need for ammonia as it transitions from flea gut to mammalian temperature. The need for ammonia might indicate increased amino acid interconversion. However, the consequences of raised intracellular ammonia concentrations are unknown.
iii. Dicarboxylic amino acids
Dicarboxylic amino acids serve a number of important roles in bacterial physiology. Aside from their role as building blocks for proteins, these molecules also act as amine donors and acceptors, as well as intracellular signaling metabolites. Furthermore, due to their facile conversion into intermediates of the TCA cycle, GLU and ASP can serve as important supplementary nutrients that provide a cell with both the carbon and nitrogen that it needs for proper growth. It has been proposed that a lesion in the metabolism of dicarboxylic amino acids might account for YPs sluggish growth. The gene aspA which encodes for the enzyme aspartase plays a prominent role in catabolism of ASP. It catalyzes the deamination of ASP into fumarate, an intermediate of the TCA cycle. Absence of this enzyme coupled with low turnover rate for some of the other enzymes involved in ASP and GLU metabolism severely limits the capacity of YP to convert these amino acids into bio-energetically useful metabolites.
Under some conditions in YP, ASP tends to be a dead-end metabolite. For example, the LCR of YP results in excretion of ASP as a by-product of catabolism of exogenous glutamate. This metabolic feature results in a loss of 4 carbon nutrients which could have entered the cellular carbon metabolism via production of fumarate and oxaloacetate. However, compensation for this nutrient loss explains the stimulatory effect of CO2 on the growth of YP. CO2 and bicarbonate can be converted into oxaloacetate through carboxylation of phosphoenolpyruvate by the enzymes phosphoenolpyruvate carboxykinase and phosphoenolpyruvate carboxylase.
Accordingly, the absence of ASPA in YP means that the primary pathway for conversion of dicarboxylic amino acids to TCA intermediates is the NADP + dependent deamination of GLU to -ketoglutarate by glutamate dehydrogenase. Therefore, although the specific activity of glutamate dehydrogenase in YP is comparable to that of YPS, the excessive metabolic burden can overwhelm the capacity of this enzyme to metabolize dicarboxylic amino acids. Additionally, use of NADP+ by glutamate dehydrogenase complicates the situation and further strains YPs metabolic mechanisms. Due to YPs deficiencies in the aerobic portion pentose phosphate pathway, the cell is forced to rely heavily on the activity of transhydrogenase enzyme to oxidize the resulting NADPH before it can be used for oxidative phosphorylation.
*Some Other Metabolisms include Nucleotide metabolism, Fatty acid metabolism, Iron Transport metabolism, and Transmission Factor metabolism. These metabolisms show no notable effects on the growth of the bacteria but have different functions and is currently being studied.
References
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