Yersinia pestis dissemination in a host is usually studied by enumerating bacteria in the tissues of animals sacrificed at different times. This laborious methodology gives only snapshots of the infection, as the infectious process is not synchronized. In this work we used in vivo bioluminescence imaging (BLI) to follow Y. pestis dissemination during bubonic plague. We first demonstrated that Y. pestis CO92 transformed with pGEN-luxCDABE stably emitted bioluminescence in vitro and in vivo, while retaining full virulence. The light produced from live animals allowed to delineate the infected organs and correlated with bacterial loads, thus validating the BLI tool. We then showed that the first step of the infectious process is a bacterial multiplication at the injection site (linea alba), followed by a colonization of the draining inguinal lymph node(s), and subsequently of the ipsilateral axillary lymph node through a direct connection between the two nodes. A mild bacteremia and an effective filtering of the blood stream by the liver and spleen probably accounted for the early bacterial blood clearance and the simultaneous development of bacterial foci within these organs. The saturation of the filtering capacity of the spleen and liver subsequently led to terminal septicemia. Our results also indicate that secondary lymphoid tissues are the main targets of Y. pestis multiplication and that colonization of other organs occurs essentially at the terminal phase of the disease. Finally, our analysis reveals that the high variability in the kinetics of infection is attributable to the time the bacteria remain confined at the injection site. However, once Y. pestis has reached the draining lymph nodes, the disease progresses extremely rapidly, leading to the invasion of the entire body within two days and to death of the animals. This highlights the extraordinary capacity of Y. pestis to annihilate the host innate immune response.
References
[1]
Yersin A (1894) La peste bubonique à Hong-Kong. Ann Inst Pasteur 662–667.
[2]
Schrag SJ, Wiener P (1995) Emerging infectious diseases: what are the relative roles of ecology and evolution? Trends Evol Ecol 10: 319–324.
[3]
Simond P (1898) La propagation de la peste. Ann Institut Pasteur 12: 625–687.
[4]
Pollitzer R (1954) Plague. World Heath Organization Monograph Series.
[5]
Chen TH, Meyer KF (1965) Susceptibility of the langur monkey (Semnopithecus entellus) to experimental plague: pathology and immunity. J Infect Dis 115: 456–464.
[6]
Guinet F, Ave P, Jones L, Huerre M, Carniel E (2008) Defective innate cell response and lymph node infiltration specify Yersinia pestis infection. PLoS One 3: e1688.
[7]
Bizanov G, Dobrokhotova ND (2007) Experimental infection of ground squirrels (Citellus pygmaeus Pallas) with Yersinia pestis during hibernation. J Infect 54: 198–203.
[8]
Hoessly GF, Walker DL, Larson A, Meyer KF (1955) Experimental Bubonic Plague in Monkeys. I. Study of the Development of the Disease and the Peripheral Circulatory Failure. Acta Trop 12: 240–251.
[9]
Sebbane F, Gardner D, Long D, Gowen BB, Hinnebusch BJ (2005) Kinetics of disease progression and host response in a rat model of bubonic plague. Am J Pathol 166: 1427–1439.
[10]
Brubaker B (2006) Yersinia pestis and Bubonic Plague. In: Dworkin M, Falkow S, Rosenberg E, Schleifer K-H, Stackebrandt E, editors. A Handbook on the Biology of Bacteria Proteobacteria: Gamma Subclass. Springer. pp. 399–442.
[11]
Francis KP, Yu J, Bellinger-Kawahara C, Joh D, Hawkinson MJ, et al. (2001) Visualizing Pneumococcal Infections in the Lungs of Live Mice Using Bioluminescent Streptococcus pneumoniae Transformed with a Novel Gram-Positive lux Transposon. Infect Immun 69: 3350–3358.
[12]
Lane MC, Alteri CJ, Smith SN, Mobley HLT (2007) Expression of flagella is coincident with uropathogenic Escherichia coli ascension to the upper urinary tract. Proc Natl Acad Sci 104: 16669–16674.
[13]
Brock M, Jouvion G, Droin-Bergere S, Dussurget O, Nicola MA, et al. (2008) Bioluminescent Aspergillus fumigatus, a New Tool for Drug Efficiency Testing and In Vivo Monitoring of Invasive Aspergillosis. Appl Environm Microbiol 74: 7023–7035.
[14]
Cook SH, Griffin DE (2003) Luciferase Imaging of a Neurotropic Viral Infection in Intact Animals. J Virol 77: 5333–5338.
[15]
Glomski IJ, Piris-Gimenez A, Huerre M, Mock M, Goossens PL (2007) Primary Involvement of Pharynx and Peyer's Patch in Inhalational and Intestinal Anthrax. PLoS Pathog 3: e76.
[16]
Bina XR, Miller MA, Bina JE (2010) Construction of a bioluminescence reporter plasmid for Francisella tularensis. Plasmid 64: 156–161.
[17]
Isaksson EL, Aili M, Fahlgren A, Carlsson SE, Rosqvist R, et al. (2009) The Membrane Localization Domain Is Required for Intracellular Localization and Autoregulation of YopE in Yersinia pseudotuberculosis. Infect Immun 77: 4740–4749.
[18]
Uliczka F, Pisano F, Kochut A, Opitz W, Herbst K, et al. (2011) Monitoring of gene expression in bacteria during infections using an adaptable set of bioluminescent, fluorescent and colorigenic fusion vectors. PLoS One 6: e20425.
[19]
Tr？ek J, Berschl K, Trülzsch K (2010) In vivo analysis of Yersinia enterocolitica infection using luxCDABE. FEMS Microbiol Lett 307: 201–206.
[20]
Russell W, Burch R (1959) The Principles of Humane Experimental Technique. London: Methuen & Co Ltd. 238 p.
[21]
Du YD, Galyov E, Forsberg A (1995) Genetic analysis of virulence determinants unique to Yersinia pestis. In: Ravagnan G, Chiesa C, editors. Yersiniosis: Present and Future. Basel: Karger. pp. 321–324.
[22]
Bland DM, Eisele NA, Keleher LL, Anderson PE, Anderson DM (2011) Novel Genetic Tools for Diaminopimelic Acid Selection in Virulence Studies of Yersinia pestis. PLoS ONE 6: e17352.
[23]
Andreu N, Zelmer A, Fletcher T, Elkington PT, Ward TH, et al. (2010) Optimisation of bioluminescent reporters for use with mycobacteria. PLoS One 5: e10777.
[24]
Rocchetta HL, Boylan CJ, Foley JW, Iversen PW, LeTourneau DL, et al. (2001) Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection. Antimicrob Agents Chemother 45: 129–137.
[25]
Henken S, Bohling J, Ogunniyi AD, Paton JC, Salisbury VC, et al. (2010) Evaluation of biophotonic imaging to estimate bacterial burden in mice infected with highly virulent compared to less virulent Streptococcus pneumoniae serotypes. Antimicrob Agents Chemother 54: 3155–3160.
[26]
Xiong YQ, Willard J, Kadurugamuwa JL, Yu J, Francis KP, et al. (2005) Real-Time In Vivo Bioluminescent Imaging for Evaluating the Efficacy of Antibiotics in a Rat Staphylococcus aureus Endocarditis Model. Antimicrobial Agents and Chemotherapy 49: 380–387.
[27]
Flexner S (1901) The pathology of bubonic plague. Am J Med Sci 122: 396–416.
[28]
Harrell MI, Iritani BM, Ruddell A (2008) Lymph node mapping in the mouse. J Immunol Methods 332: 170–174.
[29]
Jawetz E, Meyer KF (1944) The behaviour of virulent and avirulent P. pestis in normal and immune experimental animals. J Infect Dis 74: 1–13.
[30]
Frank KM, Schneewind O, Shieh W-J (2011) Investigation of a Researcher's Death Due to Septicemic Plague. N Engl J Med 364: 2563–2564.
[31]
Guarner J, Shieh WJ, Chu M, Perlman DC, Kool J, et al. (2005) Persistent Yersinia pestis antigens in ischemic tissues of a patient with septicemic plague. Human Pathol 36: 850–853.
[32]
Demeure CE, Blanchet C, Fitting C, Fayolle C, Khun H, et al. (2012) Early Systemic Bacterial Dissemination and a Rapid Innate Immune Response Characterize Genetic Resistance to Plague of SEG Mice. J Infect Dis 205: 134–143.
[33]
Flashner Y, Fisher M, Tidhar A, Mechaly A, Gur D, et al. (2010) The search for early markers of plague: evidence for accumulation of soluble Yersinia pestis LcrV in bubonic and pneumonic mouse models of disease. FEMS Immunol Med Microbiol 59: 197–206.
[34]
Parkhill J, Wren BW, Thomson NR, Titball RW, Holden MT, et al. (2001) Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413: 523–527.
[35]
Conchas RF, Carniel E (1990) A highly efficient electroporation system for transformation of Yersinia. Gene 87: 133–137.
[36]
Reed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Hyg 27: 493–497.
