Cell envelope components of Yersinia pestis grown in intraperitoneal diffusion chambers

CELL ENVELOPE COMPONENTS OF YERSINIA PESTIS GROWN IN INTRAPERITONEAL DIFFUSION CHAMBERS

Rita C. C. Ferreira^1*, Armando A. B. Neto², Sérgio E. L. Fracalanzza², Sérgio O. P. Costa¹, D. F. Almeida³, Luís C. S. Ferreira³

¹Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil; ²Instituto de Microbiologia Prof. Paulo de Góes and ³Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ, Brasil

Approved: July 23, 1998

ABSTRACT

The electrophoretic profiles of penicillin binding proteins (PBPs) and outer membrane proteins (OMPs) of Yersinia pestis EV 76 were determined following in vivo growth in diffusion chambers implanted in the peritoneal cavity of mice. In contrast to Y. pestis grown under in vitro conditions which activate the low calcium response (LCR) regulon there was no significant qualitative or quantitative change of the PBP profile of Y. pestis cells during growth in diffusion chambers for up to 72 h following implantation in mice. Three OMPs, with molecular weight of 100, 60 and 58 kDa, were expressed in Y. pestis cells grown for 24 h, but not at 48 h or at 72 h, in diffusion chambers. These results indicate that growth of Y. pestis in intraperitoneal diffusion chambers activates genes which might be relevant to the growth in the mammal host.

Key words: Yersinia pestis, PBP, in vivo growth, OMP.

INTRODUCTION

Yersinia pestis is transmitted by the bite of infected fleas to rodents and humans causing a highly invasive and fulminant disease, plague, which was probably responsible for at least 200 million deaths throughout mankind recorded history (11). Pathogenic Y. pestis strains harbor a 75 kb plasmid encoding a set of proteins and regulatory functions which are expressed in vitro at 37°C in low calcium-containing media (1). The low calcium response (LCR) involves the expression of more than 40 genes some of them encode the Yersinia outer proteins (Yop) which are essential for survival of Y. pestis in the mammalian host (1, 15). Other virulence-associated factors as the V and W antigens (16), the biosynthesis of siderophore and respective outer membrane receptors (2), and other cell envelope proteins (16) are also induced during in vitro conditions which mimic the in vivo parameters.

Recently we have shown that Y. pestis cultivated under LCR-inducing conditions suffer a gradual decrease in enzymatic activity and/or stability of several cytoplasmic membrane proteins involved in the last steps of cell wall biosynthesis, the penicillin-binding proteins (PBPs) (3,4). PBPs are found in all peptidoglycan-containing bacteria and participate in essential physiological functions of the cell envelope as maintenance of cell shape, osmotic pressure resistance, septum formation and growth in length of the bacterial cell (10). PBPs are also the lethal targets of the ß-lactams antibiotics, substrate homologues which covalently bind to them and inhibit the transpeptidase reaction performed by high molecular weight forms (17). The demonstration that Y. pestis PBPs are subjected to regulation by the same environmental conditions which trigger the LCR regulon suggested that cell wall metabolism may play an important role in pathogenicity (3). Nonetheless, an important step in the demonstration that Y. pestis PBPs may perform important functions during the infective cycle requires the study of in vivo grown cells.

The development and use of animal chamber models for in vivo growth of bacterial species have contributed significantly to the knowledge of different aspects of microbial pathogenesis (7). The chamber allows bacterial cells to be exposed to host body fluids without contacting host cells or organs. In the present study we used mice intraperitoneal diffusion chambers to efficiently recover in vivo grown Y. pestis cells. The patterns of outer membrane proteins (OMPs) and PBPs of cells grown under these conditions suggest that the implanted chamber model can be an alternativa approach for the study of genes expressed during Yersinia infection.

Bacterial strain and growth conditionsY. pestis⁶

Diffusion chambers and in vivo growth conditions. Diffusion chambers (15 mm in length and 10mm in diameter) were cut as pieces of polypropylene disposable tuberculin-type syringe canisters. Millipore 0.22 µm pore size nitrocellulose filters (Millipore, Bedford, Mass.) were cut into 1.5 mm diameter disks and stuck to the chambers ends by melting the borders and pressing them against the filters. Bacterial cells were injected in autoclaved chambers through a hole pierced on the side of the chamber with a hot needle. The hole was sealed with the help of a hot glass rod. Swiss mice weighting approximately 25 g were anesthetized with i.p. sodium pentabarbital (60 µg/g). Two chambers were placed in the peritoneal cavity through a longitudinal incision (1.5 cm) in the abdomen, which was immediately sutured. After different periods of time, the animals were sacrificed in ether-saturated atmosphere before removing the chambers. The bacterial cells were collected from the chambers to determine the number of viable cells and to isolate cell envelopes. Experiments were carried out using four mice for each time point assayed, and repeated at least twice.

Isolation of cell envelopes. Cells were harvested by centrifugation at 4°C, washed once with 50 mM sodium phosphate buffer (pH 7.0), resuspended in the same buffer, and subjected to sonic disruption with cooling. Unbroken cells were removed by low-speed centrifugation (5,000 x g for 10 min) at 4°C. The cell envelope fraction was recovered in a refrigerated microcentrifuge (20,000 x g for 1 h), suspended in phosphate buffer and the protein content was determined by the method of Lowry et al. (8).

Detection of PBPs and outer membrane proteins (OMPs). Cell envelope aliquots containing approximately 100 µg of total protein were labeled with [

Viable cell determination. Viable Y. pestis cells were determined in 50 µl aliquots removed from the implanted chambers after sacrifice of the mice. Cells were immediately diluted in PBS and plated on BHI agar plates. The number of viable cells was determined by colony counting 48 h after incubation at 28ºC.

Growth of Y. pestis EV76 in diffusion chambersY. pestis⁶Y. pestis⁵Fig. 1Y. pestis

Figure 1:
Survival curve of Y. pestis EV 76, monitored by plate counting, in diffusion chambers after implantation in the peritoneal cavity of mice. Cells were plated in BHI agar plates and incubated at 28^oC for 48 h. Surviving fractions are represented by relative values (1 = 10⁶ cells/ml).

SDS-PAGE of outer membrane-enriched fractions. Outer membrane-enriched fractions, extracted by differential solubilization of cell envelopes with Sarkosyl, of Y. pestis cells grown in diffusion chambers were analyzed by SDS-PAGE (Fig. 2). Similar OMP profiles composed by a major set of proteins with apparent molecular weights ranging from 35 to 42 kDa were observed in cells grown for greater than 24 h. Cells grown in diffusion chambers up to 24 h expressed three OMPs with molecular weights of 100, 60 and 58 kDa which were not found in cells grown in vitro (BHI) or in those kept in vivo for longer periods.

Figure 2
: SDS-PAGE (Coomassie Blue-stained) OMP profiles of Y. pestis grown in diffusion chambers implanted in the peritoneal cavity of mice. OMP profiles of cells grown for 24 h (1), 48 h (2) and 72 h (3) were determined in 10% acrylamide gels. An outer membrane-enriched fraction of Y.pestis cells grown in BHI at 28°C is also shown (C). The positions and corresponding estimated molecular weights of OMPs expressed in Y. pestis cells grown for 24 h in diffusion chambers are indicated in lane 1. Molecular weight markers are indicated on the right side of the figure.

PBP profiles of in vivo grown Y. pestis cells. The PBP patterns of Y. pestis EV76 grown in diffusion chambers for different periods were determined after labelling isolated cell envelopes with [³H]-benzylpenicillin. The PBP pattern of Y. pestis grown in vivo was composed by six major bands of 97 to 40 kDa. The same profile was found in samples removed from implanted chambers after 24 h, 48 and 72 h (Fig. 3). Such electrophoretic PBP pattern was also found in Y. pestis grown in vitro in BHI broth at 28°C. In contrast, Y. pestis grown in vivo at LCR-inducing conditions suffer a gradual decrease in the labeling of all PBPs but PBP2 (3). The present results indicate that the enzymatic activity and/or stability of PBPs in Y. pestis grown in implanted diffusion chambers or in vitro, under LCR regulon-activated conditions, are distinctly regulated.

Figure 3:
Electrophoretic profiles of the PBPs of Y. pestis EV76 grown under different conditions. Cell envelopes of Y. pestis grown in implanted diffusion chambers for 24 h (1), 48 h (2), or 72 h (3) were labeled with [3H]-benzylpenicillin and submitted to SDS-PAGE and fluorography. A cell envelope sample of Y. pestis grown in BHI at 28°C was similarly labeled and included for comparison. Radioactive molecular weight markers (M) are indicated on the left side of the figure.

DISCUSSION

Expression of genes encoding virulence-associated traits are usually regulated by specific environmental stimuli. Although some in vitro growth conditions can trigger the expression of specific virulence traits, the precise in vivo environment faced by a bacterial pathogen during infection is still largely unknown. Therefore, determination of the in vivo expression of antigens by pathogenic bacteria based on animal models is still an important approach not only for understanding the pathogenic processes but also for the development of effective vaccines and diagnostic tests. Moreover, the study of bacterial proteins or metabolic process targeted by antimicrobial agents during in vivo growth may have important consequences for antibiotic therapy.

In this work we have analyzed cell envelope proteins of Y. pestis using intraperitoneal diffusion chambers implanted in mice. We have previously shown that Y. pestis, as well as other pathogenic Yersinia, grown at 37°C in low calcium-containing medium suffer a gradual but drastic change in the enzymatic activity and/or stability of most PBPs (3,4). At the end of 36 h under such growth conditions the Y. pestis PBP profile changes from a complex pattern composed of six major proteins to a rather simple pattern composed of only PBP2 and reduced amounts of PBP5 and PBP6 (3). Our present results show that no significant change in the PBP profile of Y. pestis could be detected during growth in diffusion chambers. These data could be interpreted as a demonstration that LCR-induced conditions do not occur during growth in diffusion chambers. On the other hand, these results can indicate that signals required for in vivo activation of the LCR regulon were not generated during growth in the diffusion chambers. Blood and extracellular fluids should contain enough Ca²⁺ to repress the LCR and it has been recently shown that activation of the Yersinia LCR regulon requires bacteria-cell contact (6,12). The lack of significant proliferation of Y. pestis kept in diffusion chambers for up to 72 h suggests that such system was not reproducing the conditions found during the infection process. Therefore, the unaltered PBP profile observed in cells grown in diffusion chamber may just reflect the limitation of the system to reproduce the in vivo conditions.

Iron is a well established essential nutrient that is chelated by mammalian proteins, as lactoferrin and transferrin, making it less available to invading pathogens. To counteract host iron scavenger mechanisms, numerous other gram-negative bacteria, like Y. pestis, possesses several ferric uptake systems which are expressed during growth in iron-depleted media (2,11). OMPs induced by low iron containing media are probably the most thoroughly documented cases of in vivo expression of bacterial antigens (9,13,18). In our experiments we could observe that, 24 h following implantation of diffusion chamber into the peritoneal cavity of mice, Y. pestis cells express three prominent OMPs of 100, 60 and 58 kDa which were not detected in cells harvested at later times, when the cells were no longer engaged into active growth. Although the identities of these proteins are presently unknown, preliminary evidence indicates that they are weakly induced under iron-restricted conditions (unpublished observations). Other OMPs induced by low iron containing media have been identified in Y. pestis (2, 5, 11) but none of them match in size and relative abundance those detected now in diffusion chambers. It is thus possible that this model reveals the in vivo expression of novel proteins involved with ferric ion uptake in Y. pestis. The detection of three new OMPs in Y. pestis samples incubated for 24 h in diffusion chambers suggests that these proteins were either expressed only by actively growing cells or were required only during the initial phases of the infection.

This is the first report dealing with cell envelope proteins of Y. pestis cultivated in diffusion chambers implanted in the peritoneal cavity of mice. The usefulness of the method to identify virulence-associated factors seems to be limited, due to the lack of important bacterial-host cell contacts. Nonetheless, the possibility to identify bacterial traits which depend on physical and/or chemical factors not found under ordinary in vitro growth conditions makes this approach a tool of considerable interest for the study of Y. pestis pathogenesis.

ACKNOWLEDGMENTS

We thank the helpful technical assistance of M. A. Americo. We are also grateful to Dr. R. R. Brubaker for supplying the EV76 strain. This study was supported in part by FINEP and CNPq grants. R.C.C.F. is a recipient of a post-doctoral fellowship from the CNPq.

RESUMO

Proteínas ligadoras de penicilina em células de Yersinia pestis cultivadas in vivo

Os perfis eletroforéticos de proteínas ligadoras de penicilina (PLPs) e proteínas de membrana externa (PMEs) de Yersinia pestis EV 76 foram determinados após crescimento in vivo em câmaras de difusão implantadas na cavidade peritoneal de camundongos. Em contraste com o observado em amostras de Y. pestis crescidas in vitro em condições que ativem a resposta ao baixo nível de cálcio (RBC), não houve mudanças qualitativas ou quantitativas no perfil de PLPs de Y. pestis durante o crescimento em câmaras de difusão por até 72 h após a implantação em camundongos. Três PMEs, com peso molecular de 100, 60 e 58 kDa, foram expressas por células de Y. pestis em 24 h, mas não em 48 e 72 h, após a implantação das câmaras de difusão. Estes resultados indicam que o crescimento de células de Y. pestis em câmaras de difusão intraperitoniais podem resultar na ativação de genes relevantes para o crescimento no hospedeiro mamífero.

Palavras-chave: Yersinia pestis. PLP, crescimento in vivo growth, PME.

REFERENCES

1. Brubaker, R. R. Factors promoting acute and chronic disease caused by yersiniae. Clin. Microbiol. Rev., 4:309-324, 1991.

2. Carniel., E., D. Mazigh, and H. H. Mollaret. Expression of iron-regulated proteins in Yersinia species and their relation to virulence. Infect. Immun., 55:277-280, 1987.

3. Ferreira, R. C. C., J. T. Park, and L. C. S. Ferreira. Plasmid regulation and temperature-sensitive behavior of the Yersinia pestis penicillin-binding proteins. Infect.Immun., 62:2404-2408, 1994.

4. Ferreira, R. C. C., D. F. de Almeida and Luís C. S. Ferreira. The penicillin-binding proteins (PBPs) of the pathogenic Yersinia species. Rev. Microbiol., 27:111-115, 1996.

5. Fetherston, J. D., J. W. Lillard, Jr., and R. D. Perry. Analysis of the pesticin receptor from Yersinia pestis: role in iron-deficient growth and possible regulation by its siderophore. J. Bacteriol., 177:1824-1833, 1995.

6. Forsberg, A, R. Rosqvist, and H. Wolf-Watz. Regulation and polarized transfer of Yersinia outer proteins (Yops) involved in antiphagocytosis. Trends Microbiol., 2:14-19, 1994.

7. Genco, C. A., and R. J. Arko. Animal chamber models for study of host-parasite interactions. Methods Enzymol., 235:120-140, 1994.

8. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem., 193:265-275, 1951.

9. Morck, D. W., B. D. Ellis, P. D. G. Domingue, M. E. Olson and W. J. Costerton (1991). In vivo expression of iron-regulated outer membrane proteins in Pasteurella haemolytica-A1. Microb.Pathog., 11:373-378, 1991.

10. Park, J. T. Murein synthesis. In F. C.Neidhardt, J. L.Ingraham, K. B. Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (eds.). Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C., 1987, pp. 663-671.

11. Perry, R. D., and J. D. Fetherston. Yersinia pestis - etiologic agent of plague. Clin. Microbiol. Rev.,10:35-66, 1997.

12. Rosqvist, R., K. E. Magnusson, and H. Wolf-Watz. Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J., 13:964-972, 1994.

13. Sciortino, C. V., and R. A. Filkelstein. Vibrio cholerae express iron-regulated outer membrane proteins in vivo. Infect. Immun., 42:990-996, 1983.

14. Spratt, B. G. Properties of the penicillin-binding proteins of Escherichia coli K12. Eur. J. Biochem., 72:341-352, 1977.

15. Straley, S. C.. The low Ca²⁺ response virulence regulon of human pathogenic Yersinia. Microb. Pathog., 10:87-91, 1991.

16. Straley, S. C., and R. R. Brubaker. Cytoplasmic and membrane proteins of yersinia cultivated under conditions simulating mammalian intracellular environment. Proc. Natl.Acad. Sci. U.S.A., 78:1224-1228, 1981.

17. Waxman, D. J., and J. L. Strominger. Penicillin-binding proteins and the mechanism of action of b-lactam antibiotics. Ann. Rev. Biochem., 52:825-869, 1983.

18. Williams, P. Role of the cell envelope in bacterial adaptation to growth in vivo in infections. Biochemie, 70:987-1011, 1988.

Publication Dates

• Publication in this collection
  26 Feb 1999
• Date of issue
  Sept 1998

History

• Accepted
  23 July 1998
• Received
  09 Feb 1998
• Reviewed
  17 Apr 1998