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ИНФОРМАЦИЯ
A.Rakin
The 11th International Symposium on Yersinia: Crowdsourcing and Multilateral Cooperation - the Way to Address Bacterial and Data Challenges
Max von Pettenkofer-Institute, LMU, Munich; Germany
The 11th international Symposium on Yersinia was held in Suzhou, China June 24 - June 28, 2013. The meeting was organized by Cold Spring Harbor Asia and arranged by Ruifu Yang, Beijing Institute of Microbiology and Epidemiology, China; Elisabeth Carniel, Institut Pasteur, France; Paul Keim, Northern Arizona University, U.S.A.; and Andrey Anisimov, State Research Center for Applied Microbiology and Biotechnology, Obolensk, Russia. The Symposium covered different aspects of Yersinia physiology, epidemiology, diagnostics, infection and virulence, genomics and other Omics-driven studies. The keynote presentation by William Goldman from the University of North Carolina, U.S.A., briefly summarized the present state of Yersinia research and the impact of some outstanding scientists. The special lecture by Nils Christian Stenseth discussed the interplay between ecology and evolution in triple host-vector-pathogen systems using the plague agent, Y pestis, as an example.
One of the most fascinating lectures was done by Joe Hinnebusch who presented new data on possible mechanisms of Y. pseudotuberculosis conversion to a flea-borne transmission on its way to the plague agent. He retraced the evolutionary progression of genetic changes that might enable Y. pseudotuberculosis to build up a biofilm not in the hindut but rather in the proven-triculus of the flea, ability that is important for Y pestis midgut blockage. Addition of the Ymt phospholipase D by acquisition of the ymt gene on a transmissible pFra plasmid is sufficient to allow Y. pseudotuberculosis to colonize the midgut. However, modification of at least three other genes (rcsApe, PDE2-pe and PDE3pe) was necessary to establish stable Y. pseudotuberculosis infection in the flea’s midgut. Another enzyme, the urease, demonstrated an acute toxicity for X. cheopsis fleas by the enteropathogenic Yersinia species, Y. pseudotuberculosis and Y. enterocolitica. However the gene is silenced in the plague bacillus. Thus, silencing of the insecticidal toxicity of the urease might be an important step in Y. pseudotuberculosis adaptation to the flea-borne transmission and a significant step on its road to Y. pes-tis. Such data were presented by Imart Chokha from Joe Hinnebusch’s group.
Elisabeth Carniel (Institute Pasteur, Paris, France) continued with the characterization of the impact of genomic sequences differently represented in Y. pestis and the enteropathogenic Y. pseudotuberculosis. This time she addressed the role of a conserved RfaH regulator that activates known virulence associated genes
like hemolysin and O antigen in Escherichia coli and Salmonella Typhimurium. However, deletion of the rfaH gene in the virulent Y. pestis CO92 did not alter its ability to resist against actions of the deoxycholate, sodium dodecal sulfate and polymixin B. Moreover, the mutant retains its ability to withstand the bactericidal activity of the human serum at a same degree as the wild type strain. This greatly suspects the importance of RfaH in Y. pestis virulence. Anne Debrise from the same group applied bioluminescence imaging to trace the course of plague infection in live animals. Using the luciferase reporter and the Tn7-based transposon delivery system, they monitored the expression of six different genes (pla, yopE, ail, caf1M, psaA and fyuA) in the murine infection model of bubonic plague. They observed the in vivo sequential expression of pla, yopE, ail, caf1M, and fyuA but not of the psaA promoters from the site of subcutaneous injection to the spleen and liver, and up to terminal septicemic phase. These data once more emphasize the importance of the established virulence genes encoded by the three virulence associated plasmids (pFra, pYV and pPla), and by the High-pathogenicity island, encoding the Yersiniabactin iron acquisition system, and argue against a direct role of psaA in Y. pestis virulence. The same author reported on the construction of a live attenuated Y. pseudotuberculosis strain with an operon encoding the fraction 1 antigen stably integrated into the host chromosome. This strain V674TnF1 stably produces the Fra1 antigen and confers 100 % protection against the bubonic plague after a challenge with 103 CFU (100 x LD50) of Y. pestis. Even a single subcutaneous injection of 107 CFU of the recombinant strain protected 100 % animals against the bubonic plague (challenged with 105 CFU).
The group of Petra Dersch from the Heimholtz Centre for Infection Research, Brunswick, Germany, prepared interesting presentations on virulence regulators in Y. pseudotuberculosis. Aaron Nuss monitored temperature-dependent bistability of the pivotal virulence transcriptional global regulator RovA. Barbara Waldmann, using RNA deep sequencing, has done Y. pseudotuberculosis transcriptional profiling in different temperature conditions, at environmental (25 0C) and host (37 0C) temperatures. She identified a large number of new putative small RNAs and validated some of them by Northern blotting. Petra Dersch summarized the data in a complex model in which the nucleotide-associated thermosensitive protein YmoA, together with the Csr system (including the small regulatory RNAs CsrB and CsrC and RNA-
binding protein CsrA), represents the central part of a composite regulatory network. This network controls the switching from a RovA-dependent early colonization phase to a pYV virulence plasmid infection phase in order to escape host defenses.
Biofilm formation in Y pestis was addressed in several remarkable presentations. Joanne Purves from the University of Nottingham, United Kingdom, investigated quorum-sensing controlled genes and found that the histidine metabolic regulator HutC acts as a biofilm and motility regulator and provides a link between quorum sensing, virulence and central metabolism in Y. pseudotuberculosis. Stephan P. Willias from the University of Texas, Galveston U.S.A. showed that biofilm formation in Y. pestis was differentially modulated by the available carbon sources. While glucose inhibited biofilm production, alternative carbon sources supported the production of biofilms. The authors concluded that cAMP receptor protein CRP acts as a critical facilitator of Y. pestis and affects biofilm formation. Yi-Cheng Sun from Chinese Academy of Medical Sciences and Peking Union Medical College, China, addressed the role of two diguanylate cyclases, HmsT and Y3730, responsible for the synthesis of c-di-GMP, a cyclic diguanylate that controls biofilm formation in Y. pestis. They proposed that Y3729, that resides in the periplasm and interacts directly with the sensor domain of Y3730, senses the environmental signals and regulates the enzymatic activity of diguanylate cyclase Y3730 and c-di-GMP synthesis and thus biofilm formation.
Mikael Skurnik from the University of Helsinki, Finland, discussed the role of the surface located environmentally-exposed virulence factors: the trimeric autotransporter adhesion protein YadA, the integral adhesion and invasion outer membrane protein Ail and lipopoly-saccharide LPS with structural and endotoxin activity. He described the roles of these surface located structures in complement resistance and sensitivity to phages. Motohiro Matsuura from Kyoto University Graduate School of Medicine, Kyoto, Japan, addressed modifications of lipid A structure in Y. pestis lipopolysaccharide that facilitate bacterial evasion of the human innate immunity. The temperature changes during infection cycles from lower temperatures (external environments or in fleas) to higher temperatures (mammal host) are pretty similar in all pathogenic Yersinia. This suggests that the production of a less immunostimulatory form of the LPS during the entry into the mammalian host is conserved in Yersinia genus.
Several lectures covered different aspects of metal acquisition systems and transporters in Yersinia. Alexander Rakin from Max von Pettenkofer Institute. Munich, Germany, argued against singularity of the yers-iniabactin (Ybt) iron uptake system in highly pathogenic Yersinia species. While most of Y pseudotuberculosis O1 strains carry the functional ybt-cluster, the “outbreak” O1 strains (responsible for the Far East Scarlet-like Fever, FESF), as well as serotype O2, O4 and O5 strains are devoid of these genes, but still demonstrate a siderophore
activity. Two iron acquisition gene clusters are present in Y. pestis / Y. pseudotuberculosis group: ynp and ysu, able to encode two additional siderophores, pseudochelin and yersiniachelin, correspondently.
Jacqueline D. Fetherston from University of Kentucky, Lexington, U.S.A., addressed the role of the yfe, feo and a putative fetMP ferrous iron transporters in Y pestis. The authors demonstrated that the yfe, feo double mutants have a more severe defect in strains with deleted pgm locus than in strains containing genes both for the ybt synthesis and fetPM. They speculate that the Ybt system moderately contributes to iron acquisition under microaerobic growth conditions but does not account for all of it, while the fetMP seems to pose a stronger impact on growth in these conditions.
Robert Perry from the same University addressed the role of another metal cofactor required for the enzymatic activity of bacterial and eukaryotic cells, zinc. Although zinc is toxic at high concentrations, maintaining zinc homeostasis is essential for survival and defense of bacteria in mammalian hosts. Previously they have reported on a Y. pestis znu mutant that lacks the ZnuABC high-affinity zink uptake system. However, a deletionpgm, znu double mutant demonstrated a more pronounced growth defect in a low Zn containing medium than a znu parent strain. Moreover, the authors revealed that the double mutant is seriously attenuated in septicemic mouse model of plague compared to the parent strain with a deleted pgm locus. From this the authors concluded that the chromosomal pgm locus might encode an additional high-affinity Zn uptake system.
Virginia Miller from the University of North Carolina at Chapel Hill, U.S.A., studied the early events in bubonic plague post inoculation of Y. pestis into the skin and trafficking to the draining lymph nodes and spleen. They applied a dissemination assay with 9 nucleotide oligo tagged strains to outline a window of opportunity for plague bacillus dissemination and define whether Y. pestis passes through a bottleneck on its pathway to the spleen. They determined that only few tagged strains could be found in the spleen of the infected animals. Thus, Y. pestis indeed passes through a bottleneck on its peripheral route from skin through lymph nodes to the spleen. However, the state of Y. pes-tis population tagged with 9 nucleotide oligos and possible influence of such manipulation on the final results cannot be excluded.
An Israel group of Emanuelle Mamroud was interested in the cross talk between the lung and the bone-marrow at early stages of another plague form, a pneumonic plague. They showed that intranasal infection of mice with a fully virulent Y. pestis strain was sensed early after infection by the bone-marrow compartment, indicating a potential cross-talk between the lung and the bone-marrow already at the early stages of pneumonic plague. Both Y. pestis and soluble antigens were detected in the late stages in bone-marrow.
The group of Deborah Anderson from the University of Missouri, Columbia, U.S.A., established a pneumonic
plague model in Brown Norway Rats that closely mimics a human disease. In such model they compared the natural disease history caused in animals challenged with low (5x103) and high (5x105) Y. pestis doses within 3 days. The authors defined very high frequency of hydronephrosis, which is a common incidental finding in aged Brown Norway Rats and founded that bacteremia was not always present and nearly 40% of rats in high dose group demonstrated no clinical signs before being found dead.
Juergen Heesemann has presented interesting data on different murine innate immunity systems mechanics to Y enterocolitica ssp. enterocolitica infection. They demonstrated three different levels of susceptibility to Yersinia infection in a set of knock-out mice: highly-resistant group, wild-type resistant group and high-sus-ceptibility group. The degree of Yersinia-resistance correlated with the immigration rate of neutrophils and concentration of G-CSF, KC, IL-1a and MIP-2 within the first 24 hours post infection. The highly sensitive mice responded with a delay of 24 hours post infection compared with the highly resistant and wild-type resistant groups. Also, Yersinia invasion of the spleens of highly sensitive mice resulted in overwhelming Yersinia growth with evident destruction of the tissue architecture.
James Bliska from the Stony Brook University, NY, U.S.A., described their recent progress in understanding a dual nature of the virulence factors by studying the type 3 secretion system. They concentrated on the role of the protective epitope of the GTPase-activating protein YopE and its contribution to adaptive immunity against Y. pseudotuberculosis and possible interactions with other effectors.
Ake Forsberg from the Umea University, Sweden, described the intracellular targeting of virulence type 3 effectors in the absence of pore formation in his presentation. He presented further evidences against an out-of-date simplified model describing the T3SS mediated targeting exclusively by pore formation in the host cell membranes. He also further demonstrated the involvement of the protective LcrV antigen in the early delivery of the T3SS effectors to their respective targets.
There were multiple presentations concerning epidemiology aspects of Yersinia and Yersinia-caused diseases. Interesting data on genetic diversity, epidemiological features and distribution of pathogenic Yersinia were presented by Andrey Anisimov, from the State Research Center for Applied Microbiology and Biotechnology, Obolensk, Russia; Mark Achtman from the University College Cork, Republic of Ireland; Alzira M.P. Almeida from the Centro de Pesquisas Aggeu Magalhaes, Brazil; Minoarisoa M. Rajerison from the Institut Pasteur de Madagaskar, Madagaskar; Waldemar Rastawicki from the National Institute of Public Health-National Institute of Hygiene, Poland; Huaiqi Jing from the Chinese Center for Disease control and Prevention, Beijing, Chaina; Alan McNally from the Nottingham Trent University, United Kingdom, David M. Wagner from the Northern Arizona University, U.S.A. and in numerous poster pre-
sentations.
Several presentations dealt with paleogenetics. Holger Scholz from the Bundeswehr Institute of Microbiology, Munich, Germany, in cooperation with scientists from Germany, U.S.A. and Norway, tried to answer the question whether Y. pestis was the real agent of the Justinianic Plague of the 6th-8th centuries 541 - 767 A.D. They were able to confirm the presence of the Y. pestis sequences in the tooth samples of the human skeletal remains from the Early Medieval cemetery 6th - 7th century in Aschheim near Munich. The findings appear to confirm that Y. pestis might be really responsible for the Justinianic Plague. However, the question about how the plague arrived in southern Germany remains open.
Also Kirstin Bos from the University of Tuebingen, Germany, address a genomic reconstruction of Y. pestis from archaeological populations using next generation sequencing biased against longer fragments compared to PCR. They coupled whole genome array-based target enrichment with next generation sequencing and reconstructed Y pestis draft genome from London, England, 1348-1350. Because the authors reconstructed the ancient genome using an extant Y. pestis CO92 they were not able to identify the regions of differences that may be existed in the ancient isolate bur later lost in modern strains. However even the draft genome sequence let the researchers recognize the lack of nucleotide differences between the medieval and modern bugs. Thus the question about what contributed to high mortality of the Black Death still remains. Whether it was climate, changes in vector population, different genetic susceptibility of the host population, or con-current disease and human behavior is not known and these questions remain till now without answers. To mention, de novo reconstructed ancient Mycobacterium leprae genomes did not significantly differ from modern M. leprae strains from patient biopsies (Schuenemann V.J., et al., 2013). Thus such questions whether the ancient strains were more or less virulent or a simple improvement in sanitation results in eradication of these deadly diseases remain unanswered.
Sandra Reuter from the Wellcome Trust Sanger Institute, Cambridge, United Kingdom, together with her collaborators from Finland, France, Ireland and U.K., used whole genome sequencing approach to delineate the gene complement of Yersinia genus and define the patterns of virulence evolution. They determined genomic sequences of more than 100 strains of both pathogenic and environmental Yersinia and analyzed 85 housekeeping genes that allowed them to define more than 6000 SNPs. The authors concluded that, contrary to a popular view on pathogenic Yersinia species sharing a common pathogenic ancestor, Yersiniae have evolved independently following parallel evolutionary paths in acquiring the same virulence determinants (like independent acquisition of the pYV virulence plasmid happened on multiple occasions) and limiting their metabolic repertoire (e.g. mutations in cob/pdu, cel and ttr operons
known to be important in evolution of host specificity in Salmonella). However, a possible origin and donor of the pYV plasmid remains unclear.
The motto of the 11th international Symposium on Yersinia can be defined as “crowdsourcing and multilateral cooperation to react to the challenges of today”. This was clearly expressed by Ruifu Yang from Beijing Institute of Microbiology and Epidemiology, China. He attracted the attention of the Symposium to the necessity of creating an international consortium that will practices Open Source genomics and Crowdsourcing analysis in order to address the big data challenge by modern biological science. He mentioned that it is the right time to establish a comprehensive open database for Yersinia that includes the increasing number of genomic, transciptomic, proteomic, metabolomics, phe-nomic and interactomic data. Only cooperation of different institutions and Genome Analysis Crowdsourcing allowed fast identification and characterization of the new rapidly evolved E. coli O104:H4 clone responsible for the deadly outbreak in Germany (https://github.com/
ehec-outbreak-crowdsourced/BGI-data-analysis/wiki). Emergence of such a strain once more demonstrates the constant flexibility of bacterial genomes and necessity of wide international cooperation in combating the challenges of Mother Nature.
The next, 12th Symposium on Yersinia is supposed to take place in Tbilisi, Georgia, in three years. Looking forward to meet you in Tbilisi!
References
1. Yersinia 11: The 11th international symposium on Yersinia. Abstracts of papers presented at the 2013 Cold Spring Harbor Asia Conference, June 24 - June 28, 2013. Cold Spring Harbor Laboratory, 2013.
2. Schuenemann V.J, Singh P., Mendum T.A., Krause-Kyora B, Jager G., Bos K.I., Herbie A., Economou C., Benjak A., Busso P., Nebel A., .Boldsen J.L., Kjellstrom A., Wu H., Stewart G.R., Taylor G.M., Bauer P., Lee O.Y., Wu H.H., Minnikin D.E., Besra G.S., Tucker K., Roffey S., SowS.O., Cole S.T., NieseltK., Krause J. Genome-wide comparison of medieval and modern Mycobacterium leprae. Science. 2013; 341(6142):179-83.
Authors:
Rakin A. Max von Pettenkofer-Institute, LMU, Pettenkofer str. 9a, 80336 Munich, Germany. E-mail: rakin@mvp.uni-muenchen.de
