Y. pestis in humans
Due to biofilm blockage of the proventriculus structure in fleas, when fleas feed and intake blood, they are unable to intake the blood beyond the biofilm blockage. Resultingly, the fleas regurgitate the blood back into the bite site. However, the blood that is reentering the bitten species (i.e. humans or mammals) is now contaminated with Y. pestis.
Y. pestis requires uniquely high concentrations of calcium ions1. More specifically, Y. pestis required calcium at 37 C, but not at 27 C 2,3. When the bacteria was raised at 37 C and starved of calcium, first RNA synthesis is shut off, and next accumulation of DNA, protein, and cellular mass slows1. After RNA is significantly reduced, ATP and GTP levels begin to fall. 1. At 37 C and in calcium-deficient conditions, the bacterial cells become non-proliferative, but still viable cells.4 Y. pestis are calcium-dependent.
Type III secretion mechanism
The Type III secretion system (T3SS), though first described in Y. pestis, has been observed in plant, animal, and insect pathogens.5 These systems inject the bacterial proteins into cells and these proteins then hijack cell machinery and co-opt the host cells for their own proliferation.5,6 In Y. pestis, the system is composed of Yersinia outer membrane proteins (yops) and the ysc (yop secretion apparatus) injectisome. Unlike other secretion systems, such as the sec-mediated general secretory pathway which is the primary pathway for the secretion of extracellular degradative enzymes, the Ysc-Yop system does not require a cleavable signal peptide.7,8 In the sec-mediated secretory pathway, the mainly hydrophobic amino-terminal signal sequence aids protein export and is cleaved off by a periplasmic signal peptidase when the protein reaches the periplasm.8 The Ysc-Yop system is a T3SS; thus, it is independent of the sec system and does not involve amino-terminal cleavage, it is a continuous process that does not have periplasmic intermediates.8 In the sec-mediated system, the secretion signal is thought to be located in the amino-terminal residues of the protein, the cleavable portion. In the Ysc-Yop system, there is a high tolerance for changes in the amino-terminal residues without impacting secretion.9 Since the amino-terminal is so variable, it is unlikely that the secretion signal is located within the amino-terminal of Ysc-Yop proteins. The secretion signal might be located in the 5’region of the mRNA encoding for the secreted protein.10
The Ysc-Yop system requires approximately 20 proteins. Many of the components of the Ysc–Yop system are encoded by the pYV plasmid.11 The calcium dependency of Y. pestis cells has implications for the expression of yops, some of the components of the system.12 After incubation at 37 C in the absence of calcium, Y. pestis strains stop growing and produce large amounts of yops.12 There are two regulatory systems, a negative calcium regulatory system and a positive temperature regulation. The virF gene encodes a transcriptional activator, that controls yop expression in Y. enterocolitica, is homologous to lcrF in Y. pestis.13 The transcriptional activators turn on yop transcription at 37C, the temperature of hosts, and not at lower ambient temperatures of 26C or 30C.14
Type III Secretion System: Ysc injectisome
Most of the proteins of the Ysc-Yop system are located in the inner membrane of Y. pestis cells. Many of these proteins are homologous to components of the flagellum (Figure 1).8 Flagellum is primarily a motility organelle but serves other biological functions in the cell, including adhesion and immunity.15
The ysc injectisome secrets the yops into host cells.5 The yops have to cross several barriers before reaching the host cells; yops have to cross the inner membrane, the periplasmic space, the peptidoglycan layer, and the outer membrane of the Y. pestis cells.5 The injectisome requires direct host cell contact for activation of the secretory pathway; the injectisome must adhere to the host cell to translocate the effector proteins, such as the cytotoxic yops, into the cell.5,16
The injectisome consists of a basal body and a needle-like structure. The basal body crosses the inner membrane, the peptidoglycan layer, and the outer membrane. The needle-like structure goes outside of the Y. pestis cells.
Similar to the basal body of the flagellum, the basal body of the T3SS injectisome has an internal protein pump. The YscN protein (7.4.2.8), which resembles the catalytic subunit of protein translocase (7.4.2.3), is an essential component of the protein pump.17 Protein translocase transports proteins into the mitochondria through the Translocase of the inner membrane complex.18 Protein translocase is an ATP phosphohydrolase.17 The YscN protein is also an ATP phosphohydrolase; both catalyze the reaction in Figure 2.19,20 Through its ATPase activity the protein allows for constant incorporation of new monomers into the needle structure, thus allowing the needle to penetrate into membranes.21 Additionally, the YscN protein’s ATPase activity allows it to remove chaperones from the yop proteins.
The other component of the basal body is the YscC protein. The YscC protein forms a 20nm wide ring-shaped structure with a central pore.22 The protein forms a multimeric complex in the bacterial outer membrane and has a molecular weight of 600kDa.22 In order to correctly target the Ysc protein complex to the outer membrane, the VirG protein is required.22 The VirG product is a lipoprotein with outer membrane localization, it likely directly interacts with the YscC protein to direct the Ysc complex to the outer membrane.22
The needle of the injectisome is formed by polymerization of the YscF protein.21 The needle of the injectisomes are 60-80 nanometers (nm) long and 6-7 nm wide, and have a hollow center with a width of 2 nm.21 The needle is composed of 200-300 copies of the YscF protein.21 Since the distance between bacteria and eukaryotic cells in thin sections is less than 40 nm, the injectisome needle is more than long enough to reach eukaryptic cells and translocate the yops.21 The needle itself is necessary for hemolytic activity of Yersinia species.21 Yersinia species express up to 100 injectisome needles on their surface, the sheer amount of piercing that occurs when the bacteria come in contact with red blood cells explains the contact-dependent hemolytic activity of Y. pestis (Figure 3).21 The injectisome needle is hydrophobic, which partially explains its ability to insert itself into membranes.21
More proteins involved in forming the injectosome are yopD and yopB. The chaperone of the yopD protein is SycD (LcrH).23,24 The yopD protein has a molecular weight of 33.3kDa and has a hydrophobic domain that indicates that the protein is a transmembrane protein.24 The yopB protein is also an essential protein of the Ysc-Yop machinery. The yopB protein also uses the chaperone protein SycD (LcrH).23–25 The yopB protein is 41.8kDa protein also has two hydrophobic regions, that allow for its transmembrane location.24 These proteins help to form the central pore and guide the cytotoxic yops that are transported to the target cell.16
References
(1) Zahorchak, R. J.; Charnetzky, W. T.; Little, R. V.; Brubaker, R. R. Consequences of Ca2+ Deficiency on Macromolecular Synthesis and Adenylate Energy Charge in Yersinia Pestis. J. Bacteriol. 1979, 139 (3), 792–799.
(2) Kupferberg, L. L.; Higuchi, K. ROLE OF CALCIUM IONS IN THE STIMULATION OF GROWTH OF VIRULENT STRAINS OF PASTEURELLA PESTIS. J. Bacteriol. 1958, 76 (1), 120–121.
(3) Smith, J. L.; Higuchi, K. STUDIES ON THE NUTRITION AND PHYSIOLOGY OF PASTEURELLA PESTIS. J. Bacteriol. 1959, 77 (5), 604–608.
(4) Charnetzky, W. T.; Brubaker, R. R. RNA Synthesis in Yersinia Pestis During Growth Restriction in Calcium-Deficient Medium. J. Bacteriol. 1982, 149 (3), 1089–1095.
(5) Cornelis, G. R. The Yersinia Ysc–Yop “Type III” Weaponry. Nat. Rev. Mol. Cell Biol. 2002, 3 (10), 742. https://doi.org/10.1038/nrm932.
(6) Rosqvist, R.; Magnusson, K. E.; Wolf-Watz, H. Target Cell Contact Triggers Expression and Polarized Transfer of Yersinia YopE Cytotoxin into Mammalian Cells. EMBO J. 1994, 13 (4), 964–972.
(7) Pugsley, A. P. The Complete General Secretory Pathway in Gram-Negative Bacteria. Microbiol. Rev. 1993, 57 (1), 50–108.
(8) Hueck, C. J. Type III Protein Secretion Systems in Bacterial Pathogens of Animals and Plants. Microbiol. Mol. Biol. Rev. 1998, 62 (2), 379–433. https://doi.org/10.1128/MMBR.62.2.379-433.1998.
(9) Galán, J. E.; Collmer, A. Type III Secretion Machines: Bacterial Devices for Protein Delivery into Host Cells. Science 1999, 284 (5418), 1322–1328. https://doi.org/10.1126/science.284.5418.1322.
(10) Anderson, D. M.; Schneewind, O. A MRNA Signal for the Type III Secretion of Yop Proteins by Yersinia Enterocolitica. Science 1997, 278 (5340), 1140–1143. https://doi.org/10.1126/science.278.5340.1140.
(11) Michiels, T.; Vanooteghem, J. C.; Lambert de Rouvroit, C.; China, B.; Gustin, A.; Boudry, P.; Cornelis, G. R. Analysis of VirC, an Operon Involved in the Secretion of Yop Proteins by Yersinia Enterocolitica. J. Bacteriol. 1991, 173 (16), 4994–5009. https://doi.org/10.1128/jb.173.16.4994-5009.1991.
(12) Michiels, T.; Wattiau, P.; Brasseur, R.; Ruysschaert, J. M.; Cornelis, G. Secretion of Yop Proteins by Yersiniae. Infect. Immun. 1990, 58 (9), 2840–2849. https://doi.org/10.1128/iai.58.9.2840-2849.1990.
(13) Cornelis, G.; Sluiters, C.; de Rouvroit, C. L.; Michiels, T. Homology between VirF, the Transcriptional Activator of the Yersinia Virulence Regulon, and AraC, the Escherichia Coli Arabinose Operon Regulator. J. Bacteriol. 1989, 171 (1), 254–262. https://doi.org/10.1128/jb.171.1.254-262.1989.
(14) Yother, J.; Chamness, T. W.; Goguen, J. D. Temperature-Controlled Plasmid Regulon Associated with Low Calcium Response in Yersinia Pestis. J. Bacteriol. 1986, 165 (2), 443–447. https://doi.org/10.1128/jb.165.2.443-447.1986.
(15) Haiko, J.; Westerlund-Wikström, B. The Role of the Bacterial Flagellum in Adhesion and Virulence. Biology 2013, 2 (4), 1242–1267. https://doi.org/10.3390/biology2041242.
(16) Cornelis, G. R.; Wolf-Watz, H. The Yersinia Yop Virulon: A Bacterial System for Subverting Eukaryotic Cells. Mol. Microbiol. 1997, 23 (5), 861–867. https://doi.org/10.1046/j.1365-2958.1997.2731623.x.
(17) EC 7.4.2.3 – mitochondrial protein-transporting ATPase – BRENDA Enzyme Database https://brenda-enzymes.info/enzyme.php?ecno=7.4.2.3 (accessed 2022 -04 -29).
(18) Voos, W.; Martin, H.; Krimmer, T.; Pfanner, N. Mechanisms of Protein Translocation into Mitochondria. Biochim. Biophys. Acta BBA – Rev. Biomembr. 1999, 1422 (3), 235–254. https://doi.org/10.1016/S0304-4157(99)00007-6.
(19) EC 7.4.2.8 – protein-secreting ATPase and Organism(s) Yersinia pestis^Yersinia sp. – BRENDA Enzyme Database https://www.brenda-enzymes.org/enzyme.php?ecno=7.4.2.8&Suchword=&reference=&UniProtAcc=&organism%5B%5D=Yersinia+pestis&organism%5B%5D=Yersinia+sp.&show_tm=0 (accessed 2022 -04 -29).
(20) Wilharm, G.; Dittmann, S.; Schmid, A.; Heesemann, J. On the Role of Specific Chaperones, the Specific ATPase, and the Proton Motive Force in Type III Secretion. Int. J. Med. Microbiol. 2007, 297 (1), 27–36. https://doi.org/10.1016/j.ijmm.2006.10.003.
(21) Hoiczyk, E.; Blobel, G. Polymerization of a Single Protein of the Pathogen Yersinia Enterocolitica into Needles Punctures Eukaryotic Cells. Proc. Natl. Acad. Sci. 2001, 98 (8), 4669–4674. https://doi.org/10.1073/pnas.071065798.
(22) Koster, M.; Bitter, W.; De Cock, H.; Allaoui, A.; Cornelis, G. R.; Tommassen, J. The Outer Membrane Component, YscC, of the Yop Secretion Machinery of Yersinia Enterocolitica Forms a Ring-Shaped Multimeric Complex. Mol. Microbiol. 1997, 26 (4), 789–797. https://doi.org/10.1046/j.1365-2958.1997.6141981.x.
(23) Wattiau, P.; Bernier, B.; Deslée, P.; Michiels, T.; Cornelis, G. R. Individual Chaperones Required for Yop Secretion by Yersinia. Proc. Natl. Acad. Sci. 1994, 91 (22), 10493–10497. https://doi.org/10.1073/pnas.91.22.10493.
(24) Cornelis, G. R.; Boland, A.; Boyd, A. P.; Geuijen, C.; Iriarte, M.; Neyt, C.; Sory, M.-P.; Stainier, I. The Virulence Plasmid of Yersinia, an Antihost Genome. Microbiol. Mol. Biol. Rev. 1998, 62 (4), 1315–1352. https://doi.org/10.1128/MMBR.62.4.1315-1352.1998.
(25) Neyt, C.; Cornelis, G. R. Role of SycD, the Chaperone of the Yersinia Yop Translocators YopB and YopD. Mol. Microbiol. 1999, 31 (1), 143–156. https://doi.org/10.1046/j.1365-2958.1999.01154.x.
The regulation of the injectisome by temperature and calcium is fascinating! Is the concentration of calcium needed for the organism’s survival and to overcome feedback inhibition of the injectisome similar to the normal serum calcium concentration in humans? Are there additional modulators or regulators of the complex like host cytokines or markers of hemolysis like bilirubin or LDH?
Hi Lindsey! I learned a lot about the pathogenesis of Y. Pestis from reading your blog! You mention that “The injectisome requires direct host cell contact for activation of the secretory pathway.” As you probably know, bacterial adhesion to host cells is an important step during infection. I am wondering, for Y. pestis, is direct contact of the injectisome alone responsible for mediating this interaction? What are some of the recognition sites on healthy cells that stabilize the binding event?
Although direct contact of the injectisome is essential for mediating the interaction between Y. pestis and the host cell, there are other recognition sites that are important.
The paper by Cornelis in 2002 suggested that contact between the membrane lipids allows for the secretion mechanism. The paper also suggested that adhesins are likely involved. (1)
The paper by Chauhan et al. from 2016 reviews the many adhesin proteins involved in the infection process. Some adhesin genes present and their functions (if known) in Y. pestis are:
-YapA (unknown function)
-YapC (binding to epithelial cells and macrophages and for biofilm formation)
-YapE (binding to eukaryotic cells, autoaggregation)
-YapF-N (unknown function)
-YapV (interacts with actin-polymerizing factor)
-YadB (promotes survival in skin after flea bite)
-YadC (promotes survival in skin after flea bite)
-InvC/Ilp (adhesion to and invasion of host cells)
-Psa/Myf (binding to galactose and biofilm formation)
-Caf (protection from phagocytosis and binding to interleukin-1B)
-y0348–0352 (adhesion to macrophages)
-y0561–0563 (biofilm formation)
-y1858–1862 (adhesion to macrophages)
-y1869–1873 (adhesion to macrophages)
-y2388–2392 (unknown function)
-y3478–3480 (unknown function)
The specific mechanism of adhesion to macrophages is not known for many of these adhesins.
(2)
(1) Chauhan, N.; Wrobel, A.; Skurnik, M.; Leo, J. C. Yersinia Adhesins: An Arsenal for Infection. PROTEOMICS – Clinical Applications 2016, 10 (9–10), 949–963. https://doi.org/10.1002/prca.201600012.
(2) Cornelis, G. R. The Yersinia Ysc–Yop “Type III” Weaponry. Nature Reviews Molecular Cell Biology 2002, 3 (10), 742. https://doi.org/10.1038/nrm932.