Resistance of Plaque Pathogen to Innate Immunity
Serum and Cationic Antimicrobial Peptide Resistance in Isogenic Lipopolysaccharide Mutants of Yersinia pestis
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
3 Approved without Funding
Institute of Organic Chemistry, Russia, Moscow
- State Research Center for Applied Microbiology and Biotechnology, Russia, Moscow reg., Obolensk
- Forschungszentrum Borstel / Leibniz-Zentrum für Medizin und Biowissenschaften, Germany, Borstel\nUniversity of Illinois / College of Medicine, Rockford, USA, IL, Rockford\nUniversitat de Barcelona, Spain, Barcelona\nHarvard Medical School / Brigham and Women's Hospital, USA, MA, Boston
Project summaryThe innate immunity is of central importance in the host defense against bacteria intruding into the animal organism. The only way for pathogenic bacteria to protect themselves against the immune defenses of the healthy person is expression of a set of bacterial offensive and defensive pathogenicity factors (Finlay, Falkow, 1997). One of these is the lipopolysaccharide (LPS), a component of the outer membrane, located on the cell surface of Gram-negative bacteria (Raetz, Whitfield, 2002). It displays multifarious activities responsible for both stimulation and overcoming the immunity. The full, smooth-type LPS consists of lipid A, a core oligosaccharide, and an O-polysaccharide chain (O-antigen) but some pathogenic bacteria, including Yersinia pestis, the cause of plague (Chart et al., 1995, Hitchen et al., 2002, Prior et al., 2001a, Skurnik et al., 2000, Vinogradov et al., 2002), have a short-chain, rough-type LPS. In Y. pestis, the lack of the O-antigen is a result of inactivation of the O-antigen gene cluster by frame-shift mutations (Prior et al., 2001b, Skurnik et al., 2000).
LPS structure variations can nullify the immune response ensuring that infections are not recognized fully or efficiently by the host immune memory. For instance, temperature dependent variations in the LPS of Y. pestis, namely the production at 37°C of a lower acylated lipid A portion, likely results in a less immunostimulatory LPS, which may compromise the host’s ability to rapidly respond with a proper inflammatory response to infection (Kawahara et al., 2002, Rebeil et al., 2004). The LPS structure also mediates resistance of Y. pestis to complement-mediated lysis (serum resistance) (Porat et al., 1995) that is necessary for survival and growth in mammal blood to be transmitted between its insect and mammalian hosts (Brubaker, 2000, Domaradskii, 1993, Perry, Fetherston, 1997, Anisimov, 1999, 2002a, 2002b, Perry, 2003). Furthermore, the Y. pestis LPS structure determines bacterial resistance to cationic antimicrobial peptides (CAMP) (Bengoechea et al., 1998), a key component of the innate immunity in both mammals and insects (Dimopoulos, 2003).
Recently, in the framework of the ISTC project # 1197, we performed a comprehensive analysis of the Y. pestis LPS structure, including lipid A and core structures, and studied its interrelation with the susceptibility to NHS and polymyxin B (PMB) as a model of CAMP in various subspecies of Y. pestis of different geographical occurrence and epidemiological significance cultivated at mammalian (37C), flea (25C) or winter-hibernation (6C) temperatures. In ssp. pestis (main subspecies) high levels of resistance to PMB were detected at 25C. However, Y. pestis strains of ssp. hissarica, caucasica, and fresh isolates of ssp. altaica were highly sensitive to PMB at the same temperature. At 6C and 37C, all strains were highly susceptible to PMB. In contrast to other strains, at 25C and 37C strains of ssp. caucasica were highly susceptible to the bactericidal activity of 80% NHS, whereas all Y. pestis strains were able to grow in heat-inactivated NHS or in 80% normal mouse serum. At 6C, all strains were highly sensitive to NHS. The temperature-induced variations in biological properties of different bacterial cultures were found to be related, in part, with changes in the LPS structures (Knirel et al., 2005a, 2005b, Anisimov et al., 2005). Particularly, it was suggested that both high content of 4-amino-4-deoxyarabinose in lipid A and a proper combination of terminal monosaccharides in the core achieved at 25C are important for CAMP resistance. NHS resistance correlated with an elevated content of N-acetylglucosamine in the core.
Our studies of virulence of Y. pestis strains from various subspecies showed that strains of all subspecies are highly lethal for mice; the overwhelming majority of ssp. pestis isolates are also lethal for guinea pigs, whereas strains of ssp. caucasica, hissarica, and altaica are of low virulence or even avirulent in guinea pigs (Anisimov et al., 2004).
The main goal of the present project is elucidation in a genetically defined background of the impact of different structural components of Y. pestis LPS on resistance to innate immunity factors, including serum and CAMPs, as well as on the activity of plasminogen activator (Pla) - another important virulence factor of the bacterium - by using isogenic Y. pestis mutants with impaired LPS biosynthesis pathway. In addition, the potential of the isogenic mutants as live vaccines will be estimated. As LPSs are immunogenic and show the species/strain/phase variant specificity, the structurally characterized LPS of different Y. pestis intraspecies groups and different isogenic mutants will be used for construction of glycoarray for differential detection of anti-Y. pestis LPS antibodies.
The tasks of the project are:
- Generation of isogenic deletion mutants with defects in different steps of LPS biosynthesis by the site-directed lambda Red recombination technology. If necessary, disruption of genes of interest by mini-transposon Tn5 mutagenesis and construction of trans-complemented strains, including strains complemented with homologous Y. pestis genes. Comprehensive characterization of the genes involved in LPS biosynthesis in search for molecular targets for new antimicrobial agents.
- Studies of biological properties of the mutants, including resistance to killing by both 80% NHS and PMB, using bacteria grown at 25°C, i.e. under the conditions in which the parent strain is highly resistant. Testing of the Pla activity in the mutant strains in order to unravel its correlation with the LPS structure.
- Estimation of the level of virulence attenuation and the potential of the mutants as live vaccines by studies of the susceptibility of the mutant bacteria to killing by phagocytes and their ability to induce TNF- in macrophage cell lines.
- Determination of the fine LPS structure in the mutants for elucidation of its correlation with the biological properties and for glycoarray construction. For this purpose, LPS will be isolated from bacterial cells of each mutant, purified and analyzed by chemical methods, high-resolution NMR spectroscopy and mass spectrometry before and after cleavage to the lipid A and core components.
The expected results will include:
- Data on the interrelatedness of Y. pestis LPS structural organization and biological properties in regard to the sensitivity to serum and CAMPs.
- Data on the natural strategies that Y. pestis uses for overcoming host innate immunity.
- Molecular targets for the design of new high-affinity inhibitors as likely efficient antimicrobial agents of new generation for therapy of plague.
- Structurally characterized Y. pestis LPS antigens for construction of glycoarray potentially useful in serodiagnostics and seroepidemiology of plague.
Participants of the project are experienced in both genetic manipulations on the model of Y. pestis and studying structural organization of bacterial LPSs (relevant publications are listed in Part II, Section 12). Only attenuated Y. pestis strains will be used in this study: EV line NIIEG (Δpgm; ssp. pestis bv. orientalis), 1146pPst?pCad?pFra? (ssp. caucasica bv. antiqua), KIM#D1 (pCad?; ssp. pestis bv. medievalis) as well as their derivatives, which will be specially generated and deposited in the culture collection of the SRCAMB. Microbiological work will be performed in the BSL2 facilities.
Achievement of the project goals will result in an essential contribution in the fields of applied biology and medicine as well as into solutions of significant social problems such as the control of bioterrorism. The project will allow the scientists and engineers of the SRCAMB, engaged previously in biodefense studies, to reorient their scientific interests and to use their prior experience to carry out fundamental and applied biomedical studies. Ultimately the project will not only serve peaceful purposes but also create long-term prospects for fruitful activity based on international cooperation and meetings with scientists through presentations at conferences. The project will provide an opportunity to seek continued international research collaboration including creation in the SRCAMB of the WHO Collaborating Centre for Research of Emerging and Reemerging Bacterial Diseases.
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