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Respiratory Disease Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, Mailstop G03, Atlanta, GA 30333, USA
Correspondence
Claressa E. Lucas
CHL9{at}cdc.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Gao et al., 1998). L. pneumophila can also parasitize protozoa in a similar, but not identical, manner (Fields, 2002; Gao et al., 1997). Legionellae and protozoa inhabit biofilms in building water systems, providing a reservoir of contamination that can persist for decades (Cowgill et al., 2005; Fields et al., 2002). The overall structure of the biofilm, in addition to providing physical protection, is a unique ecosystem in which heterogeneous species interact in a multicellular manner (Romling et al., 1998; Romling, 2001; Shapiro, 1998). Colonization, microcolony formation and maturation are distinct phases of biofilm development that require a collaborative effort from each constitutive organism, with stage-specific expression of several molecular components (Dalton & March, 1998; Le Thi et al., 2001). Environmental signals may regulate which organisms can colonize a surface as well as determine their release into the planktonic milieu (Stanley & Lazazzera, 2004).
For example, the expression of type IV pili is essential to colonization and maturation of the Pseudomonas aeruginosa biofilm on a variety of surfaces (O'Toole & Kolter, 1998). These adhesins, expressed by numerous Gram-negative micro-organisms, are characterized by sequence conservation of the structural subunits, assembly machinery, and mechanisms of transcriptional regulation (Strom & Lory, 1993). In P. aeruginosa the twitching or gliding motility associated with type IV pili are necessary for surface translocation allowing microcolony foundation (O'Toole & Kolter, 1998). These pili also participate in cell–cell communication both with related species and with diverse unrelated micro-organisms in multicellular matrices (Dorr et al., 1998; Kallstrom et al., 1998; Wall & Kaiser, 1999). Type II secretion, the mechanism by which most Gram-negative organisms export pilus machinery for assembly, also translocates several virulence factors that may contribute to biofilm maturation (China & Goffaux, 1999; Liles et al., 1999).
In order to determine whether these molecular components are essential for biofilm formation in L. pneumophila, colonization and retention of pilus mutants were examined using the biofilm model described by Murga et al. (2001). The rotating disc reactor they designed is unique in that it seeks to effectively mimic conditions in building water systems, using a moderate water flow rate, tap water rather than rich medium, and a surface (stainless steel) in common use for potable water delivery systems. Their work demonstrated that L. pneumophila could colonize an established biofilm consisting of P. aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. In the absence of a protozoan host for intracellular replication, L. pneumophila was able to persist for at least 15 days, although no growth was evident. The addition of amoebae into this system allowed for growth of L. pneumophila, providing a model for L. pneumophila persistence in building water systems.
Many Gram-negative organisms have an absolute requirement for type IV pili and the type II secretion system in order to establish a biofilm and allow for maturation (Bechet & Blondeau, 2003; Entcheva-Dimitrov & Spormann, 2004; Giltner et al., 2006; Moorthy & Watnick, 2004). However, L. pneumophila is a facultative intracellular parasite of a broad range of free-living protozoa, and thus may have evolved different mechanisms for the biofilm niche compared to free-living Gram-negative bacteria. This study investigated the ability of L. pneumophila to colonize and persist in the Murga model when altered in the expression of type IV pili or the type II secretion system. Strain NU243 contains a kanamycin resistance (KmR) cassette inserted in pilD that abolishes expression of the pre-pilin peptidase, PilD. PilD cleaves the leader sequence of proteins, such as the major type IV pilus subunit, PilE, which are exported via the type II secretion mechanism. Thus, this strain fails to express both type IV pili and type II secretion products (Liles et al., 1999). The second mutant, BS100, contains a KmR insertion in pilE that eliminates expression of the major pilin subunit, leaving type II secretion unaffected (Stone & Abu Kwaik, 1998).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Liles et al., 1999; Stone & Abu Kwaik, 1998). The heterotrophic Gram-negative bacteria used to form the biofilm matrix were Pseudomonas aeruginosa (ATCC 770), Klebsiella pneumoniae (DMDS#92-08-28a) and a Flavobacterium strain (‘F. xanthomonas’ CDC-65). The protozoan component was the amoeba Hartmannella vermiformis (ATCC 50237) (Fields et al., 1990). All bacteria were grown from frozen stocks of not more than three passages on artificial media within the laboratory.
The medium for L. pneumophila was buffered charcoal yeast extract (BCYE) with antibiotic supplement. The formulation is, per litre: 18.3 g BCYE agar base (BBL), 2 g glycine, 10 g L-cysteine, 80 mg cycloheximide, 13.22 mg polymixin B and 5 mg vancomycin. L. pneumophila was incubated for 5–7 days at 37 °C with 5 % (v/v) CO2. The heterotrophs were grown on R2A agar (Difco) at 25 °C for 3 days. The amoebae were grown in axenic medium at 37 °C and subcultured at 3–4 day intervals to prevent encystment, according to standard protocols (Fields et al., 1990). The formulation for axenic medium is, per litre: 10 g peptone and yeast extract, 1 g yeast DNA, 15 mg folic acid, 1 mg haemin, 500 mg dibasic anhydrous sodium phosphate, 362 mg monobasic anhydrous potassium phosphate and 10 % (v/v) fetal bovine serum.
Biofilm reactor and sampling methods.
The biofilm reactor has been described elsewhere (Murga et al., 2001). Briefly, 1x108 c.f.u. of each base organism were added to the reactor and allowed to form a biofilm on a disc rotator containing 316L stainless steel coupons (Biosurface Technologies) in a 1 : 10 dilution of R2A broth for 3 days at 30 °C. R2A broth consisted of, per litre: 500 mg each of yeast extract, proteose peptone #3, Casamino acids and glucose; 300 mg each of sodium pyruvate and dibasic potassium phosphate; and 50 mg magnesium sulfate. After the initial incubation period, R2A broth diluted 1 : 100 was connected for constant flow of 1 ml min–1 for 24 h followed by de-chlorinated tap water for the remainder of the experiment. Retention time under constant flow was 8.3 h.
One week after beginning the flow of tap water 1x108 c.f.u. of L. pneumophila and 4x106 cells of H. vermiformis, where applicable, were added to a reactor (day 0). Samples from both the bulk liquid and stainless steel coupons were taken on days 1, 4, 7, 11 and 16, then collected in polystyrene tubes to inhibit adherence by the amoebae. Samples of the bulk liquid were taken by pipette through a portal in the lid of each reactor. Coupons were aseptically removed from the biofilm rotator, rinsed three times in Butterfield buffer (425 mg l–1 monobasic potassium phosphate, pH 7.2) then deposited in 10 ml 10 mM PBS pH 7.2. The coupons were alternately vortexed for 30 s then sonicated for 30 s for three repetitions (setting #18 on a 60 Sonic Dismembrator, Fisher Scientific) to remove the biofilm from the surface. The collection liquid was then homogenized for 30 s (PRO2000 homogenizer, PRO Scientific) to yield a suspension of single cells.
Viable counts of heterotrophic bacteria were determined by plating serial dilutions on appropriate artificial media. L. pnemophila colonies were enumerated by first treating the sample with an acidic solution of potassium chloride for 15 min to reduce the viability of heterotrophs and amoebae (DeLuca et al., 1999). The legionellae were then deposited onto a 0.2 µm filter (Millipore Isopore Membrane GTTP) that was transferred to a tube containing sterile distilled, de-ionized water. Serial dilutions were plated from this suspension after vortexing for 1 min. Viable counts of amoebae were determined as previously described (Bowman et al., 1996) by placing 100 µl of serial dilutions from the original bulk or coupon sample on non-nutrient agar plates spread with Escherichia coli DH5
followed by incubation at 37 °C for 5 days. Microscopy was used to follow trophic spread, thereby determining the limiting dilution. This method provides only the order of magnitude of trophic amoebae present in the system and does not directly measure trophs or cysts. All data presented herein represent the means of at least four independent experiments. Statistical analysis of points compared was done with paired Student's t tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In contrast, the pilE mutant strain, BS100, attached at a significantly lower level (P<0.01). Retention of 130b and NU243 declined over time, with NU243 undetectable on day 11. BS100 and 130b were recovered throughout the the experiment at levels similar to their respective attachment. There was a slight but statistically insignificant (P>0.1) increase in 130b recovery on day 16.
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). There was no significant shedding of legionellae from the biofilm. On day 16 there was a slight increase in detection of 130b coincidental with the increased viable counts recovered from the coupons (Fig. 1a). However, the increase, when compared to the two mutant strains, is statistically insignificant (P>0.1) due to the high standard deviation. Viable counts of the bacteria that formed the base biofilm were also determined beginning 1 day before the addition of the L. pneumophila and continuing throughout the experiment. The ratio of P. aeruginosa to K. pneumoniae to ‘F. xanthomonas’ was approximately 3 : 3 : 1 both on the coupons and in the bulk water. The base biofilm bacteria were unaffected by the addition of L. pneumophila, remaining at a constant level of approximately 108 c.f.u. per coupon and 105 c.f.u. ml–1 in the bulk water regardless of the strain of L. pneumophila added (data not shown).
Biofilms in the presence of amoebae
L. pneumophila and the amoeba, H. vermiformis, were added to the reactor without prior mixing after the bacteria of the base biofilm had been allowed to establish a biofilm for 10 days. Strains 130b and BS100 colonized the established biofilm in a similar manner (Fig. 2a). Both strains displayed a steady increase of approximately 2 logs over 16 days and did not appear to have reached a plateau.
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) and was thus expected to disappear from the biofilm in a similar way to what was observed in the absence of amoebae (Fig. 1a). No NU243 were recovered on day 1 but over 102 c.f.u. per coupon were detected on day 4, identical to the wild-type and simple pilin mutant (Fig. 2a). However, NU243 did not increase after day 4, but declined slightly to a steady state of roughly 5x102 c.f.u. per coupon on day 16.
In the bulk water, strains 130b and BS100 were present from day 1 at roughly 103 c.f.u. ml–1. Viable counts for both strains peaked on day 4 at roughly 5x103 c.f.u. ml–1 then declined, to a final concentration of 102 c.f.u. ml–1 on day 16 (Fig. 2b). Note that the planktonic counts of both 130b and BS100 declined as sessile counts increased (compare Fig. 2a and 2b). NU243 was undetectable on day 1 but increased to almost 102 c.f.u. ml–1 on day 4, remaining at approximately this level until day 16.
The ratio of P. aeruginosa to K. pneumoniae to ‘F. xanthomonas’ remained approximately 3 : 3 : 1 for both sessile and planktonic fractions. Addition of amoebae reduced the base biofilm total counts from 108 c.f.u. per coupon to 105 c.f.u. per coupon and 106 c.f.u. ml–1 in the planktonic to 104 c.f.u. ml–1 (data not shown). Amoebae from the biofilm increased by one order of magnitude between days 1 and 4 to plateau at approximately 102 cells per coupon regardless of the strain of L. pneumophila introduced. All reactors contained roughly 102 cells ml–1 in the planktonic fraction on days 1–16 (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). L. pneumophila has several uncharacterized pili as well as other cell surface adhesins (Stone & Abu Kwaik, 1998, 1999). Thus, it is possible that the severe mutation in NU243 which destroys several tiers of protein expression may trigger overexpression of a PilD-independent adhesin. Further study of the cell surface adhesion molecules of legionellae is needed to clarify these results.
The action of a putative secondary mechanism of attachment did not correlate with retention of L. pneumophila. NU243 could establish itself in the biofilm at a level similar to wild-type but did not persist past day 9. This suggests that colonization and retention are distinct phases requiring expression of different cell surface components or extracellular proteins. The PilD pre-pilin peptidase is essential for the secretion of numerous degradative enzymes required for pathogenesis (Aragon et al., 2000; Cianciotto, 2001). These data imply a role for PilD-dependent secretory products in the retention of L. pneumophila in a biofilm, as well.
The presence of a protozoan host is more critical to colonization and retention than the presence of type IV pili or type II secretion. The wild-type and the PilE– mutant displayed statistically identical colonization and retention profiles in the presence of amoebae. This is in direct contrast to the greatly reduced attachment of the PilE– mutant in the absence of amoebae. Inclusion of amoebae was also able to partially compensate for the decreased retention of the PilD– mutant strain. The increase of NU243 from undetectable levels on day 1 to 102 on day 4 indicates that these observations are most likely due to intracellular replication. Furthermore, it would seem that undefined factors in the biofilm are able to partially restore the PilD– mutant's ability to replicate intracellularly.
Unlike other Gram-negative organisms commonly studied in biofilm models L. pneumophila does not have an absolute requirement for type IV pili or type II secretion. Its unique lifestyle as a facultative intracellular parasite may diminish its need for primary attachment and retention mechanisms. In the absence of protozoa there are obviously mechanisms other than pilin expression functioning to allow colonization and keep the legionellae within the biofilm. Characterization of the methods utilized by this organism for attachment and persistence in the biofilm will require further investigation.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Aragon, V., Kurtz, S., Flieger, A., Neumeister, B. & Cianciotto, N. P. (2000). Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila. Infect Immun 68, 1855–1863.
Bechet, M. & Blondeau, R. (2003). Factors associated with the adherence and biofilm formation by Aeromonas caviae on glass surfaces. J Appl Microbiol 94, 1072–1078.[CrossRef][Medline]
Bowman, E. K., Vass, A. A., Mackowski, R., Owen, B. A. & Tyndall, R. L. (1996). Quantitation of free-living amoebae and bacterial populations in eyewash stations relative to flushing frequency. Am Ind Hyg Assoc J 57, 626–633.[Medline]
China, B. & Goffaux, F. (1999). Secretion of virulence factors by Escherichia coli. Vet Res 30, 181–202.[Medline]
Cianciotto, N. P. (2001). Pathogenicity of Legionella pneumophila. Int J Med Microbiol 291, 331–343.[CrossRef][Medline]
Cowgill, K. D., Lucas, C. E., Benson, R. F., Chamany, S., Brown, E. W., Fields, B. S. & Feikin, D. R. (2005). Recurrence of Legionnaire's disease at a hotel in the United States Virgin Islands over a 20-year period. Clin Infect Dis 40, 1205–1207.[CrossRef][Medline]
Dalton, H. M. & March, P. E. (1998). Molecular genetics of bacterial attachment and biofouling. Curr Opin Biotechnol 9, 252–255.[CrossRef][Medline]
DeLuca, G., Stampi, S., Lezzi, L. & Zanetti, F. (1999). Effect of heat and acid decontamination treatments on the recovery of Legionella pneumophila from drinking water using two selective media. New Microbiol 22, 203–208.[Medline]
Dorr, J., Hurek, T. & Reinhold-Hurek, B. (1998). Type IV pili are involved in plant-microbe and fungus-microbe interactions. Mol Microbiol 30, 7–17.[CrossRef][Medline]
Engleberg, N. C., Drutz, D. J. & Eisenstein, B. I. (1984). Cloning and expression of Legionella pneumophila antigens in Escherichia coli. Infect Immun 44, 222–227.
Entcheva-Dimitrov, P. & Spormann, A. M. (2004). Dynamics and control of biofilms of the oligotrophic bacterium Caulobacter crescentus. J Bacteriol 186, 8254–8266.
Fields, B. S. (2002). The social life of legionellae. In Legionella, pp. 135–142. Edited by R. Marre and others. Washington, DC: American Society for Microbiology.
Fields, B. S., Nerad, T. A., Sawyer, T. K., King, C. H., Barbaree, J. M., Martin, W. T., Morrill, W. E. & Sanden, G. N. (1990). Characterization of an axenic strain of Hartmannella vermiformis obtained from an investigation of nosocomial legionellosis. J Protozool 37, 581–583.[Medline]
Fields, B. S., Benson, R. F. & Besser, R. E. (2002). Legionella and Legionnaires' disease: 25 years of investigation. Clin Microbiol Rev 15, 506–526.
Gao, L. Y., Harb, O. S. & Abu Kwaik, Y. (1997). Utilization of similar mechanisms by Legionella pneumophila to parasitize two evolutionarily distant host cells, mammalian macrophages and protozoa. Infect Immun 65, 4738–4746.[Abstract]
Gao, L. Y., Stone, B. J., Brieland, J. K. & Abu Kwaik, Y. (1998). Different fates of Legionella pneumophila pmi and mil mutants within macrophages and alveolar epithelial cells. Microb Pathog 25, 291–306.[CrossRef][Medline]
Giltner, C. L., van Schaik, E. J., Audette, G. F., Kao, D., Hodges, R. S., Hassett, D. J. & Irvin, R. T. (2006). The Pseudomonas aeruginosa type IV pilin receptor binding domain functions as an adhesin for both biotic and abiotic surfaces. Mol Microbiol 59, 1083–1096.[CrossRef][Medline]
Kallstrom, H., Islam, M. S., Berggren, P. O. & Jonsson, A. B. (1998). Cell signaling by the type IV pili of pathogenic Neisseria. J Biol Chem 273, 21777–21782.
Le Thi, T. T., Prigent-Combaret, C., Dorel, C. & Lejeune, P. (2001). First stages of biofilm formation: characterization and quantification of bacterial functions involved in colonization process. Methods Enzymol 336, 152–159.[Medline]
Liles, M. R., Edelstein, P. H. & Cianciotto, N. P. (1999). The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila. Mol Microbiol 31, 959–970.[CrossRef][Medline]
Moorthy, S. & Watnick, P. I. (2004). Genetic evidence that the Vibrio cholerae monolayer is a distinct stage in biofilm development. Mol Microbiol 52, 573–587.[CrossRef][Medline]
Murga, R., Forster, T. S., Brown, E., Pruckler, J. M., Fields, B. S. & Donlan, R. M. (2001). Role of biofilms in the survival of Legionella pneumophila in a model potable-water system. Microbiology 147, 3121–3126.
O'Toole, G. A. & Kolter, R. (1998). Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol 30, 295–304.[CrossRef][Medline]
Romling, U. (2001). Genetic and phenotypic analysis of multicellular behavior in Salmonella typhimurium. Methods Enzymol 336, 48–59.[Medline]
Romling, U., Sierralta, W. D., Eriksson, K. & Normark, S. (1998). Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter. Mol Microbiol 28, 249–264.[CrossRef][Medline]
Shapiro, J. A. (1998). Thinking about bacterial populations as multicellular organisms. Annu Rev Microbiol 52, 81–104.[CrossRef][Medline]
Stanley, N. R. & Lazazzera, B. A. (2004). Environmental signals and regulatory pathways that influence biofilm formation. Mol Microbiol 52, 917–924.[CrossRef][Medline]
Stone, B. J. & Abu Kwaik, Y. (1998). Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells. Infect Immun 66, 1768–1775.
Stone, B. J. & Abu Kwaik, Y. (1999). Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili. J Bacteriol 181, 1395–1402.
Strom, M. S. & Lory, S. (1993). Structure-function and biogenesis of the type IV pili. Annu Rev Microbiol 47, 565–596.[CrossRef][Medline]
Wall, D. & Kaiser, D. (1999). Type IV pili and cell motility. Mol Microbiol 32, 1–10.[CrossRef][Medline]
Received 26 July 2006;
revised 5 September 2006;
accepted 7 September 2006.
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