1 Department of Microbiology, University of Wisconsin-La Crosse, La Crosse, WI 54601, USA
2 Department of Chemistry, University of Wisconsin-La Crosse, La Crosse, WI 54601, USA
Correspondence
William R. Schwan
schwan.will{at}uwlax.edu
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In a quest to find new potential targets for the development of anti-staphylococcal drugs, a signature-tagged mutagenesis (STM) system was adapted for use in S. aureus (Coulter et al., 1998). From this STM system, one of the in vivo-attenuated mutants that were found had a mutation in the high-affinity proline transport gene putP (Schwan et al., 1998
). An external source of proline is needed by S. aureus strain RN6390; thus proline transporters, such as PutP, are critical for the survival of these bacteria. This proline auxotrophy could be a host adaptation by S. aureus strains living on warm-blooded animals. To rectify this proline auxotrophy, S. aureus has at least two proline transport systems: a low-affinity system presumably linked to a proP gene homologue and a high-affinity proline uptake system encoded by the putP gene (Bae & Miller, 1992
; Pourkomailian & Booth, 1994
; Townsend & Wilkinson, 1992
; Wengender & Miller, 1995
). Of the two proline uptake systems, the high-affinity system encoded by putP has been more thoroughly characterized. The putP gene of S. aureus was first identified by Wengender & Miller (1995)
and the STM study has shown the effects of mutating the putP gene (Schwan et al., 1998
). This putP mutant strain of S. aureus strain RN6390 was shown to have reduced proline uptake compared to the parental strain. Moreover, marked in vivo attenuation was noted in several animal models of infection, including murine systemic, wound and abscess models (Schwan et al., 1998
), and a rabbit endocarditis model (Bayer et al., 1999
; Schwan et al., 1998
).
The purpose of this study was to elucidate more thoroughly why the mutation in the high-affinity proline transport gene in S. aureus led to the marked attenuation in murine models of infection. By examining growth in low-proline media and analysing proline concentrations in murine organ tissues, we showed that the putP mutant is crippled in environments where there are low proline concentrations. Moreover, complementation of the putP mutations restored proline uptake as well as virulence.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasmids.
The plasmids used were pERL3 501/253 (Schwan et al., 1994), pPWC-1 (provided by Karen Miller, Penn State University; Wengender & Miller, 1995
), pUCGM1 (provided by Herbert Schweizer, Colorado State University; Schweizer, 1993
) and pCL84 (provided by Chia Lee, University of Kansas Medical Center). All of the plasmids were isolated from E. coli or S. aureus strains using the QIAprep spin mini-prep plasmid isolation kit (Qiagen). For plasmid isolation from S. aureus, the protocol outlined in the kit was modified. After addition of the suspension buffer, the bacterial cells were treated with 100 µg ml1 of recombinant lysostaphin (AMBI UK) at 37 °C for 10 min before the addition of the lysis buffer. The lysostaphin was used to break apart the cell wall cross-linking to allow the DNA to more readily exit from the bacterial cells.
Transfer of the putP : : Tn917 mutation to strain RN4220.
Because another background strain besides strain RN6390 was desired, the putP mutation in 16F-157 (derived from strain RN6390) was transferred to strain RN4220 via transduction (Kloos & Pattee, 1965) with a lysate of S. aureus phage
80
(provided by Ambrose Cheung, Dartmouth Medical School) from strain 16F-157 (putP : : Tn917). Transductants were selected for on trypticase soy agar (Difco) containing 2 mM sodium citrate and erythromycin. Erythromycin-resistant transductants were screened for a disruption of the putP gene by PCR. One hundred picomoles of primers SaputP5 (5'-GATGCTACCTAAAGCTAGACG-3') and SaputP6 (5'-TCTTCCGTTAGTGAACTAGATG-3'), which flank the spot where the Tn917 inserted into the genome of strain 16F-157, were used in the PCR amplifications under the following conditions: an initial denaturation at 95 °C for 5 min followed by 32 cycles of 95 °C, 1 min; 57 °C, 1 min; and 72 °C, 1 min. The absence of a 260 bp band would signify an insertion within the putP gene, whereas its presence would signify an uninterrupted gene.
Construction of the putP-complementing plasmid.
The backbone plasmid used for the complementing plasmid was pERL3 501/253, which contains an erythromycin-resistance gene, a pAM1 origin of replication that functions in many Gram-positive bacteria, including S. aureus (Luchansky et al., 1988
), and a pBR322 origin of replication for E. coli. This plasmid DNA was cut with the restriction endonuclease PstI and then blunted using T4 DNA polymerase (Boerhinger Mannheim). A gentamicin-resistance gene was isolated from pUCGM1 by cutting the plasmid DNA with SmaI, separating the fragments on a low-melting-point agarose gel, cutting out the fragment with the gentamicin-resistance gene, and processing the DNA as previously described (Schwan et al., 1992
). A ligation reaction was set up using T4 DNA ligase (New England Biolabs) to ligate the gentamicin-resistance gene isolated from pUCGM1 with the PstI-blunted pERL3 501/253 DNA. The ligation mixture was transformed into E. coli S17·1
pir (Sambrook et al., 1989
) and plated onto LB agar plates containing gentamicin, resulting in the construction of pERLGM. Following isolation of the plasmid DNA, pERLGM was cut with EcoRI and BamHI, separated on a low-melting-point agarose gel, and the appropriate fragment to be used was isolated and processed as described above. pPWC-1 DNA was cut with EcoRI and BamHI, the fragments separated on a low-melting-point gel, the putP-containing fragment cut out, and the full-length putP DNA fragment processed as noted above. A ligation was set up between the EcoRI/BamHI-cut pERLGM DNA and the isolated putP gene. The ligation mixture was transformed into E. coli strain DH5
and cells were plated onto LB agar plates containing gentamicin, resulting in the construction of plasmid pERLGMputP-19. The pERLGMputP-19 DNA was cut with XbaI and then blunted by filling in the ends by the use of E. coli Klenow fragment (Boerhinger Mannheim) (Sambrook et al., 1989
). To obtain the tetracycline-resistance gene, pCL84 DNA was cut with the restriction endonuclease HindIII. The DNA fragment with the tetracycline-resistance gene was then blunted with Klenow fragment and isolated on a low-melting-point agarose gel. Blunted pERLGMputP-19 DNA was ligated to the blunted tetracycline-resistance gene from pCL84. This ligation mixture was transformed into E. coli DH5
and plated onto LB agar plates containing gentamicin and tetracycline, resulting in construction of pTGCOMP (Fig. 1
).
|
Proline transport assay.
Transport of radioactive proline into the staphylococcal cells was measured using the filtration method described by Bae & Miller (1992). Bacteria were suspended to a final concentration of 2540 mg total cellular protein ml1 as determined with the Bio-Rad protein assay kit (Bradford, 1976
). The S. aureus cells were preincubated at 37 °C for 5 min in the transport buffer, and L-[2,3-3H]proline (Dupont/NEN) was added at a final concentration of 5 µM (specific activity, 40 Ci mmol1; 1480 GBq mmol1). The assay was set up as previously noted (Schwan et al., 1998
).
Determination of proline concentrations in murine organ tissues.
Livers and spleens were obtained from BALB/c mice (Harlan Laboratories) and were immediately frozen until the samples could be dehydrated. Dehydrated tissue samples were weighed and ground to a fine powder with a mortar and pestle. A measured amount of tissue sample (1550 mg) was hydrolysed with 6 M HCl at 110 °C for 24 h. The HCl-digested samples were neutralized with an equal amount of 6 M NaOH. Analysis for L-proline was performed by Scientific Research Consortium (St Paul, MN, USA). The absorbance at 440 and 570 nm was measured after post-column colour development by ninhydrin reagent at 131 °C using a Beckman amino acid analyser model 7300 and computations calculated with Beckman System Gold 8.10 chromatography software.
LD50 determinations.
LD50 determinations (Reed & Meunch, 1938) were performed using BALB/c mice (Harlan Laboratories) inoculated with 10-fold serial dilutions (0·5 ml) of strain RN6390, strain 16F-157 or strain 16F-157/pTGCOMP suspended in TSB containing 1 % (w/v) Nutrex administered intraperitoneally. A total of five mice per dilution were used with input titres covering a range of 102 to 108 c.f.u. ml1. LD50 values were calculated relative to the virulent wild-type parent strain RN6390.
Statistics.
Student's t-test was used to calculate statistical variation.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Testing of proline uptake in recombinant RN4220putP cells
To determine if the putP recombinant plasmid delivered into the RN4220putP cells could restore proline uptake in these defective cells, a radioactive proline transport assay was performed. Wild-type strain RN4220 cells were able to transport approximately threefold more radioactive proline than the mutant strain RN4220putP (P<0·0099; Fig. 2a). However, the putP mutation was overridden by the pTGCOMP plasmid, containing a full-length putP gene, which brought the level of proline uptake in the RN4220putP/pTGCOMP cells up to the wild-type level through the entire 10 min time-course that was studied. When strain RN6390 and the corresponding putP mutant strain 16F-157 were tested over a 10 min period, proline uptake was initially about twofold higher for the wild-type strain RN6390 versus the putP mutant strain 16F-157 (although the difference narrowed slightly during the assay period), showing that the same effect was occurring in a different background (Fig. 2b
). Complementation of strain 16F-157 also restored the proline uptake to wild-type levels.
|
|
|
Virulence of the putP mutant strain restored with the putP recombinant plasmid
As shown above, proline transport and accumulation were fully re-established by the putP recombinant plasmid. Of more interest though was whether this complementing plasmid could restore virulence to the S. aureus cells. Transposon insertions into the genome are known to create polar mutations that affect downstream genes, and these, rather than the point of insertion in one particular gene, may actually cause the phenotype. If a mutation is truly complemented, then virulence will be restored in the mutant strain. To test whether virulence was restored by the addition of the putP recombinant plasmid, LD50 determinations were run with the wild-type parental strain RN6390, its Tn917 mutant 16F-157 (putP : : Tn917) and 16F-157 bearing the putP recombinant plasmid pTGCOMP. The results showed an LD50 of 8·13x106 bacteria for the wild-type strain RN6390, which was about 10-fold lower than the LD50 of the putP mutant strain 16F-157 (7·35x107). With the transfer of the putP recombinant plasmid into the 16F-157 cells, the LD50 returned to a near wild-type level of 9·23x106. This demonstrated that the putP gene alone was needed for both full proline uptake and virulence and that complementation of the putP mutation not only restored proline uptake and growth in low-proline medium, but also the virulence of the bacterial cells.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have demonstrated that the reductions in proline transport and bacterial virulence were indeed due to the mutation of the putP gene and not some polar effect caused by the insertion of Tn917. Transformation of both the RN4220putP and 16F-157 S. aureus mutants with the putP recombinant pTGCOMP plasmid re-established optimal proline uptake that matched that of the respective wild-type parental strains, compared to the significantly lower proline uptake levels observed for each putP mutant strain. Although proline uptake was diminished in the putP mutant strains, a significant amount of proline was still taken up by the staphylococcal cells, presumably due to at least one other proline transport system in S. aureus (Bae & Miller, 1992; Townsend & Wilkinson, 1992
; Pourkomailian & Booth, 1994
). For the first time, we have also demonstrated that mutation in putP does in fact have an effect on transport in addition to the potential transport and accumulation effects that were previously measured (Bae & Miller, 1992
; Wengender & Miller, 1995
; Schwan et al., 1998
) and also confirmed in this study (Fig. 2
). A very short time-course of 15 or 30 s showed that the putP mutant strains had significantly less uptake of proline than the wild-type bacteria or the complemented strains. By using this very brief exposure assay, we have conclusively shown that there is an effect on transport because there is not sufficient time to accumulate the radioactive proline during this time-course. After only 15 s, there is an eightfold difference between the wild-type strain RN4220 and the mutant strain RN4220putP, but this ratio decreases to less than a twofold difference by 10 min, probably because of accumulation effects. This is an important distinction for us to fully understand proline transport in S. aureus. It is possible that the putP mutation may be tied to a deficiency in the transport of another entity in addition to proline. Certainly, sodium levels may be modulated because the PutP protein is thought to be a sodium co-transporter. Another explanation could be the presence of at least one other proline transport system that may be bringing proline into the staphylococcal cells. No genetic analyses have been done to investigate the other system. Nonetheless, when the proline level is low in the environment, the high-affinity proline transporter appears to be very important for bringing proline into the cell. Differences in proline uptake and accumulation within the two staphylococcal strain backgrounds studied were similar for both wild-type strains, but the putP mutant strains for each background displayed slightly different kinetic profiles over the time-frame studied.
Of more critical interest is how this defect in proline transport carries through to attenuation of a putP mutant in animal models of infection. Since S. aureus is an auxotroph for proline (Gladstone, 1937), it must import this key amino acid. The growth studies in minimal medium shed some light on why proline permeases may be a critical part of S. aureus survival. In a low-proline environment, the presumption would be that the high-affinity proline transporter would be crucial. Indeed, growth of the S. aureus putP mutant in minimal medium with 1·74 µM proline was significantly reduced compared to wild-type bacteria. Low proline levels could be triggering transcriptional activation of the putP gene that may be leading to increased PutP protein expression in the S. aureus cells growing in proline-depleted environments. When these in vitro observations are tied to the level of proline in some murine organs, the attenuation in the in vivo survival of the S. aureus makes sense. Very few studies have examined the level of amino acids, like proline or proline betaine, in murine and human fluids or tissues (Chambers & Kunin, 1987
; Inoue et al., 1996
, 1999
). Human serum has been observed to have 29·6 mM proline (Inoue et al., 1996
) and human urine has been shown to have proline concentrations that range from 6·08 to 17·10 µM (Inoue et al., 1999
). Our previous study indicated a substantial decrease in the bacterial numbers from spleens and livers infected with a putP mutant strain compared to a wild-type strain of S. aureus (Schwan et al., 1998
). Spleens and livers were targeted in this study because of the lower growth by the proline uptake mutant. No information has been previously reported for murine liver and spleen proline levels. In this study, an entire spleen of a mouse was shown to have only 7·5 µmol proline and a murine liver only 88·4 µmol proline. Certainly, proline levels should be higher in areas of the spleen and liver fed directly by the circulatory system, where higher levels of proline prevail (Inoue et al., 1996
). However, other areas of both organs should have substantially lower levels of proline that are further from direct contact with the bloodstream. Staphylococcal cells in these organs will have access to only a portion of this proline and are therefore likely to experience low proline concentrations.
The last piece of the puzzle is tying together the concept of growth in a proline-deprived environment and the virulence of the S. aureus bacterial cells. When one considers that S. aureus can infect virtually every tissue in the human body (Easmon & Adlam, 1983), one can appreciate why the putP mutant strain grew poorly in murine livers and spleens (Schwan et al., 1998
) if there is limited availability of proline at each of these sites within the mouse. Complementation of the 16F-157 putP mutant with the putP recombinant plasmid re-established the virulence of the bacteria to a near wild-type level. A study of proline transport in E. coli showed that a mutation in the low-affinity proline transport gene caused a 100-fold reduction in E. coli recovered from the bladders of the infected mice compared to wild-type E. coli with an intact proP gene (Culham et al., 1998
). These previous findings in E. coli combined with our confirmatory study here in S. aureus would suggest that proline transport is important for in vivo survival in certain niches within the animal host and presumably within the human body because of the low availability of proline in some of the tissues and bodily fluids.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bayer, A. S., Coulter, S. N., Stover, C. K. & Schwan, W. R. (1999). Impact of the high-affinity proline permease gene (putP) on the virulence of Staphylococcus aureus in experimental endocarditis. Infect Immun 67, 740744.
Boyce, J. M. (1997). Epidemiology and prevention of nosocomial infections. In The Staphylococci in Human Disease, pp. 309328. Edited by K. B. Crossley & G. L. Archer. New York: Churchill Livingstone.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72, 248254.[CrossRef][Medline]
Centers for Disease Control & Prevention (1997). Staphylococcus aureus with reduced susceptibility to vancomycin United States, 1997. Morbid Mortal Weekly Rep 46, 765766.[Medline]
Centers for Disease Control & Prevention (1999). Four pediatric deaths from community acquired methicillin-resistant Staphylococcus aureus Minnesota and North Dakota, 19971999. Morbid Mortal Weekly Rep 48, 707710.
Chambers, S. T. & Kunin, C. M. (1987). Isolation of glycine betaine and proline betaine from human urine. J Clin Invest 79, 731737.[Medline]
Coulter, S. N., Schwan, W. R., Ng, E. Y. & 7 other authors (1998). Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments. Mol Microbiol 30, 393404.[CrossRef][Medline]
Culham, D. E., Dalgado, C., Gyles, C. L., Mamelak, D., MacLellan, S. & Wood, J. M. (1998). Osmoregulatory transporter ProP influences colonization of the urinary tract by Escherichia coli. Microbiology 144, 91102.[Abstract]
Durack, D. T. & Beeson, P. B. (1972). Experimental bacterial endocarditis. II. Survival of bacteria in endocardial vegetations. Br J Exp Pathol 53, 5053.[Medline]
Easmon, C. S. F. & Adlam, C. (1983). Staphylococci and Staphylococcal Infections. New York: Academic Press.
Gladstone, G. P. (1937). The nutrition of Staphylococcus aureus: nitrogen requirements. Br J Exp Pathol 19, 208226.
Iandolo, J. J. & Kraemer, G. R. (1990). High frequency transformation of Staphylococcus aureus by electroporation. Curr Microbiol 21, 373376.
Inoue, H., Date, Y., Kohashi, K., Yoshitomi, H. & Tsuruta, Y. (1996). Determination of total hydroxyproline and proline in human serum and urine by HPLC with fluorescence detection. Biol Pharm Bull 19, 163166.[Medline]
Inoue, H., Iguchi, H., Kono, A. & Tsuruta, Y. (1999). Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labeling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride. J Chromatography 724, 221230.
Kauffman, C. A. & Bradley, S. F. (1997). Epidemiology of community-acquired infection. In The Staphylococci in Human Disease, pp. 287308. Edited by K. B. Crossley & G. L. Archer. New York: Churchill Livingstone.
Kloos, W. E. & Pattee, P. A. (1965). Transduction analysis of the histidine region in Staphylococcus aureus. J Gen Microbiol 39, 195207.[Medline]
Luchansky, J. B., Muriana, P. M. & Klaenhammer, T. R. (1988). Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium. Mol Microbiol 2, 637646.[Medline]
NNIS (1996). National Nosocomial Infections Surveillance (NNIS) report, data summary from October 1986April 1996, issued May 1996. A report from the National Nosocomial Infections Surveillance (NNIS) System. Am J Infect Control 24, 380388.[Medline]
Novick, R. P. (1990). The Staphylococcus as a molecular genetic system. In Molecular Biology of the Staphylococci, pp. 140. Edited by R. P. Novick. New York: VCH Publishers.
Panlilio, A. L., Culver, D. H., Gaynes, R. P., Banerjee, S., Henderson, T. S., Tolson, J. S. & Martone, W. J. (1992). Methicillin-resistant Staphylococcus aureus in U. S. Hospitals, 19751991. Infect Control Hosp Epidemiol 13, 582586.[Medline]
Pourkomailian, B. & Booth, I. R. (1994). Glycine betaine transport by Staphylococcus aureus: evidence for feedback regulation of the activity of the two transport systems. Microbiology 140, 31313138.[Abstract]
Reed, L. J. & Meunch, H. (1938). A simple method of estimating fifty per cent endpoints. Am J Hyg 27, 493497.
Rudin, L., Sjostrom, J. E., Lindberg, M. & Philipson, L. (1974). Factors affecting competence for transformation in Staphylococcus aureus. J Bacteriol 118, 155164.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schwan, W. R., Seifert, H. S. & Duncan, J. L. (1992). Growth conditions mediate differential transcription of fim genes involved in phase variation of type 1 pili. J Bacteriol 174, 23672375.[Abstract]
Schwan, W. R., Demuth, A., Kuehn, M. & Goebel, W. (1994). Phosphatidylinositol-specific phospholipase C from Listeria monocytogenes contributes to intracellular survival and growth of Listeria innocua. Infect Immun 62, 47954803.[Abstract]
Schwan, W. R., Coulter, S. N., Ng, E. Y. W. & 7 other authors (1998). Identification and characterization of the PutP proline permease that contributes to in vivo survival of Staphylococcus aureus in animal models. Infect Immun 66, 567572.
Schweizer, H. P. (1993). Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. BioTechniques 15, 831834.[Medline]
Sieradzki, K., Roberts, R. B., Haber, S. W. & Tomasz, A. (1999). The development of vancomycin resistance in a patient with methicillin-resistant Staphylococcus aureus infection. N Engl J Med 340, 517523.
Smith, T. L., Pearson, M. L., Wilcox, K. R. & 8 other authors (1999). Emergence of vancomycin resistance in Staphylococcus aureus. N Engl J Med 340, 493501.
Townsend, D. E. & Wilkinson, B. J. (1992). Proline transport in Staphylococcus aureus: a high affinity system and a low affinity system involved in osmoregulation. J Bacteriol 174, 27022710.[Abstract]
Wengender, P. A. & Miller, K. J. (1995). Identification of a PutP proline permease gene homolog in Staphylococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli. Appl Environ Microbiol 61, 252259.[Abstract]
Received 15 August 2003;
revised 1 December 2003;
accepted 17 December 2003.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |