Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lin, H.-H. Articles by Chen, Y.-L. Search for Related Content PubMed PubMed Citation Articles by Lin, H.-H. Articles by Chen, Y.-L.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, December 2006, p. 4596-4597, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01531-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Presence of the exoU Gene of Pseudomonas aeruginosa Is Correlated with Cytotoxicity in MDCK Cells but Not with Colonization in BALB/c Mice

Hsi-Hsun Lin,¹ Shiao-ping Huang,² Hsiu-Chen Teng,³ Dar-Der Ji,⁴ Yao-Shen Chen,⁵ and Ya-Lei Chen^6*

Department of Infectious Disease, E-DA Hospital/I-Shou University, Kaoshiung, Taiwan,¹ Department of Medical Technology, Fooyin University, Kaoshiung, Taiwan,² Department of Healthcare Management, Fooyin University, Kaoshiung, Taiwan,³ Division of Research and Diagnosis, Center for Disease Control, Taipei, Taiwan,⁴ Section of Infectious Disease, Kaoshiung Veterans General Hospital, Kaoshiung, Taiwan,⁵ Department of Biotechnology, National Kaoshiung Normal University, Kaoshiung, Taiwan⁶

Received 25 July 2006/ Returned for modification 13 September 2006/ Accepted 6 October 2006

Top Abstract Text References

 
A total of 141 independent strains of Pseudomonas aeruginosa with different heterogeneities in the exo gene (exoS, exoT, exoU, and exoY) background were examined for their pathogenic roles. Results indicated that the exoU gene is the major contributor to cytotoxicity in Madin-Darby canine kidney cells but is not related to bacterial colonization in mice.

Top Abstract Text References

 
Pseudomonas aeruginosa harbors at least one or more exoS, exoT, exoU, and exoY genes that are translated into protein products related to type III secretion systems (TTSS). These products have been demonstrated to show a cytotoxic effect in vitro. Furthermore, in clinical studies, the presence of these toxins is associated with a dissatisfactory clinical outcome among patients with P. aeruginosa infection (6, 7, 9, 10). However, these TTSS-related toxin genes show heterogeneity in terms of their presence in the genome of individual P. aeruginosa strains. For example, the presence of exoU and that of exoS are mutually exclusive (2, 4, 5). Normally, more than 90% of clinical strains contain exoT and exoY genes, but clinical isolates, if they are isolated from urine, frequently have the exoY gene present at a relatively lower level—about 70% of strains (4, 5). Due to the complicated interactions that seem to occur during TTSS-related toxin gene expression, there is uncertainty about what the presence of these various different toxin genes means in terms of P. aeruginosa strain toxicity and intrinsic strain virulence (11). In this study, we examined which TTSS-related toxicity effectors are associated with cytotoxicity against mammalian cells or are involved in bacterial colonization of the spleen and liver of animals.

The isolates of P. aeruginosa were collected from different sources, either bacteremic patients (n = 100) or environments (n = 41; including water towers or conduits), in Kaoshiung, Taiwan. Their identification as P. aeruginosa was performed by biochemical characterization using an automatic system (BD Phoenix 100 Automated Microbiology System; Becton, Dickinson and Company, Franklin Lakes, NJ). Chromosomal DNA from each isolate was extracted using a DNeasy Miniprep kit (QIAGEN Co. Ltd., Chuo-Ku, Tokyo, Japan) and then used as a template for PCR. All isolates were demonstrated to be independent based on random amplified polymorphic DNA profiling (3). This profiling was analyzed using 6% polyacrylamide gel electrophoresis and Molecular Analyst fingerprinting software (Bio-Rad, Hercules, CA). The banding patterns were tested for reproducibility by profiling them in duplicate, and an independent strain was identified when two or more amplified bands were distinct from each other. The genotype in terms of TTSS genes was determined by amplifying specific TTSS amplicons using the methods of Ajayi et al. (1).

Madin-Darby canine kidney (MDCK) epithelial cell cytotoxicity was determined by the release of lactate dehydrogenase (LDH) into the supernatant. Once the MDCK cells (10⁵/well) had attached to 24-well plastic trays, the wells were washed three times and the culture medium replaced by fetal calf serum-free Dulbecco's modified Eagle's medium. After a 1-h incubation, individual bacterial isolates (10⁷ CFU/ml) were added to the wells. After another 3 h, the concentration of LDH was quantified using a cytotoxicity kit according to the manufacturer's instructions (Sigma). The proportion of cells lysed (percentage) in a sample well was calculated as follows: {[(LDH activity in sample well) – (spontaneous release)]/[(maximum amounts of LDH release) – (spontaneous release)]} x 100.

To determine the colonization of P. aeruginosa in vivo, BALB/c mice (7 to 8 weeks old) were inoculated via the tail vein with 10⁶ CFU of a series of representative P. aeruginosa strains suspended in sterile phosphate-buffered saline (50 µl). At 18 h postinfection, the inoculated mice were killed and their livers and spleens excised. These organs were weighed and separately homogenized in sterile phosphate-buffered saline, and then serial dilutions were plated on LB agar. The CFU were determined on the next day.

MDCK cell lysis was found to be correlated with the presence or absence of the exoU (P < 0.001) or exoY (P < 0.001) gene in the P. aeruginosa isolates after a 3-h treatment, but the exoS gene showed a reverse correlation (P < 0.001). After consideration of the relationship between the different exo genes and clinical/environmental isolates by multiple regression analysis, the levels of cytotoxicity were found to be significantly related to the presence of the exoU gene (ß = 0.595; P = 0.024), more than that of the exoY gene (ß = 0.241; P = 0.001). No significant effect was found for the presence of the exoS gene (P = 0.946). In our collection of 141 isolates, 5 TTSS-related toxin genotypes were present, namely exoT⁺ exoU⁺, exoT⁺ exoU⁺ exoY⁺, exoS⁺ exoT⁺, exoS⁺ exoT⁺ exoY⁺, and exoS⁺ exoT⁺ exoU⁺ exoY⁺. On comparing the interaction of each individual gene within the exo gene complex, the mean cytotoxic effect of P. aeruginosa strains with the genotype exoT⁺ exoU⁺ exoY⁺ (42.8% ± 10.0%) was greater than that of exoS⁺ exoT⁺ exoY⁺ (29.2% ± 6.7%) or exoS⁺ exoT⁺ (24.7% ± 6.2%) strains (P < 0.001). The 18.1% difference between exoT⁺ exoU⁺ exoY⁺ strains and exoS⁺ exoT⁺ strains was higher than that between exoT⁺ exoU⁺ exoY⁺ strains and exoS⁺ exoT⁺ exoY⁺ strains (13.6%), suggesting that the exoU gene is the "major gene" and that the exoY gene does contribute to some extent to MDCK cytotoxicity caused by P. aeruginosa (Table 1).

View this table: [in this window] [in a new window]

TABLE 1. Comparison of cytotoxic effects of bacteria on MDCK cells and of bacterial colonization in BALB/c mice

BALB/c mice mimicking bacteremia were used to estimate the colonizing ability of P. aeruginosa with or without the exoU gene in vivo. Subsequent to tail injection with randomized representative isolates having the genotype exoT⁺ exoU⁺, exoT⁺ exoU⁺ exoY⁺, exoS⁺ exoT⁺, or exoS⁺ exoT⁺ exoY⁺, the concentration of P. aeruginosa in the blood was determined to be <10 CFU/ml but bacterial colonization of the spleen and liver was detectable. When the bacterial loads in the spleen and liver were correlated with the various TTSS genotypes, no significant difference was found (Table 1). The results indicated that the genes producing the TTSS-related toxins did not seem to facilitate bacterial colonization after infection with P. aeruginosa.

Schulert et al. reported that the P. aeruginosa strains isolated from hospital-acquired pneumonia patients were highly virulent if the isolate harbored the exoU gene versus isolates that did not (11). Shaver and Hauser reported that isogenic strains containing the exoU gene had greater virulence against BALB/c mice than strains containing the exoS or exoT gene (12). This effect was not synergistic if P. aeruginosa secreted the ExoU protein in combination with the ExoS or ExoT protein (13). However, P. aeruginosa causes a variety of serious infections involving multiple virulence determinants and accessory factors (8). Multiple processes, such as bacterial colonization, target site invasion, and cytotoxicity, determine the establishment of a P. aeruginosa infection. With 141 independent strains, we have demonstrated that the presence or absence of the exoU gene alone or the exoU gene together with other TTSS-related toxin genes supports the hypothesis that the exoU gene is the major contributor to potential virulence. However, the presence of exoU genes did not act as an indicator for enhanced bacterial loads in the spleen or liver in BALB/c mice. It is likely that other accessory factors alone or combined with the exoU gene were involved in bacterial colonization. The complicated interaction of multiple genes might mask a significant role in the predisposition that the exoU⁺ strains of P. aeruginosa may have to cytotoxicity, which may be relevant for bacterial colonization in vivo. Whether the exoU gene plays a role in virulence after bacterial colonization by P. aeruginosa still needs to be studied.

 
This project was supported by an NSC (ROC) grant, NSC93-2626-B-242-002-CC3.

 
* Corresponding author. Mailing address: Department of Biotechnology, National Kaoshiung Normal University, Kaoshiung, Taiwan. Fax: 886-7-615-1353. Phone: 886-7-605-1366. E-mail: dan1001{at}ma31.hinet.net.

Published ahead of print on 18 October 2006.

Top Abstract Text References

 

1
1. Ajayi, T., L. R. Allmond, T. Sawa, and J. P. Wiener-Kronish. 2003. Single-nucleotide-polymorphism mapping of Pseudomonas aeruginosa type III secretion toxins for development of a diagnostic multiplex PCR system. J. Clin. Microbiol. 41:3526-3531.[Abstract/Free Full Text]
2
2. Berthelot, P., I. Attree, P. Plesiat, J. Chabert, S. de Bentzmann, B. Pozzetto, and F. Grattard. 2003. Groupe d'Etudes des Septicemies a Pseudomonas aeruginosa. Genotypic and phenotypic analysis of type III secretion system in a cohort of Pseudomonas aeruginosa bacteremia isolates: evidence for a possible association between O serotypes and exo genes. J. Infect. Dis. 188:512-518.[CrossRef][Medline]
3
3. Campbell, M., E. Mahenthiralingam, and D. P. Speert. 2000. Evaluation of random amplified polymorphic DNA typing of Pseudomonas aeruginosa. J. Clin. Microbiol. 38:4614-4615.[Abstract/Free Full Text]
4
4. Feltman, H., G. Schulert, S. Khan, M. Jain, L. Peterson, and A. R. Hauser. 2001. Prevalence of type III secretion genes in clinical and environmental isolates of Pseudomonas aeruginosa. Microbiology 147:2659-2669.[Abstract/Free Full Text]
5
5. Ferguson, M. W., J. A. Maxwell, T. S. Vincent, J. da Silva, and J. C. Olson. 2001. Comparison of the exoS gene and protein expression in soil and clinical isolates of Pseudomonas aeruginosa. Infect. Immun. 69:2198-2210.[Abstract/Free Full Text]
6
6. Finck-Barbancon. V., J. Goranson, L. Zhu, T. Sawa, J. P. Wiener-Kronish, S. M. Fleiszig, C. Wu, L. Mende-Mueller, and D. W. Frank. 1997. ExoU expression by Pseudomonas aeruginosa correlates with acute cytotoxicity and epithelial injury. Mol. Microbiol. 25:547-557.[CrossRef][Medline]
7
7. Hauser, A. R., E. Cobb, M. Bodi, D. Mariscal, J. Valles, J. N. Engel, and J. Rello. 2002. Type III protein secretion is associated with poor clinical outcomes in patients with ventilator-associated pneumonia caused by Pseudomonas aeruginosa. Crit. Care Med. 30:521-528.[CrossRef][Medline]
8
8. Kipnis, E., T. Sawa, and J. Wiener-Kronish. 2006. Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med. Maladies Infect. 36:78-91.[Medline]
9
9. Roy-Burman, A., R. H. Savel, S. Racine, B. L. Swanson, N. S. Revadigar, J. Fujimoto, T. Sawa, D. W. Frank, and J. P. Wiener-Kronish. 2001. Type III protein secretion is associated with death in lower respiratory and systemic Pseudomonas aeruginosa infections. J. Infect. Dis. 183:1767-1774.[CrossRef][Medline]
10
10. Sato, H., and D. W. Frank. 2004. ExoU is a potent intracellular phospholipase. Mol. Microbiol. 53:1279-1290.[CrossRef][Medline]
11
11. Schulert, G. S., H. Feltman, S. D. Rabin, C. G. Martin, S. E. Battle, J. Rello, and A. R. Hauser. 2003. Secretion of the toxin ExoU is a marker for highly virulent Pseudomonas aeruginosa isolates obtained from patients with hospital-acquired pneumonia. J. Infect. Dis. 188:1695-1706.[CrossRef][Medline]
12
12. Shaver, C. M., and A. R. Hauser. 2004. Relative contributions of Pseudomonas aeruginosa ExoU, ExoS, and ExoT to virulence in the lung. Infect. Immun. 72:6969-6977.[Abstract/Free Full Text]
13
13. Shaver, C. M., and A. R. Hauser. 2006. Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals. Microbiology 152:143-152.[Abstract/Free Full Text]

---

Journal of Clinical Microbiology, December 2006, p. 4596-4597, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01531-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Lin, H.-H. Articles by Chen, Y.-L. Search for Related Content PubMed PubMed Citation Articles by Lin, H.-H. Articles by Chen, Y.-L.