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Penicillin tolerance in Streptococcus pneumoniae , autolysis and the Psa ATP‐binding cassette (ABC) manganese permease

1999; Wiley; Volume: 32; Issue: 4 Linguagem: Inglês

10.1046/j.1365-2958.1999.01369.x

1365-2958

Jean‐Pierre Claverys, Chantal Granadel, Anne M. Berry, James C. Paton,

Neonatal Health and Biochemistry

Sir, Recently, Novak et al. (1998, Mol Microbiol29: 1285–1296) reported their investigation on the phenomenon of penicillin tolerance in Streptococcus pneumoniae. A library of mutants in pneumococcal surface proteins was screened for the ability to survive in the presence of 10 × the minimum inhibitory concentration of antibiotic. A mutant harbouring an insertion in the known gene psaA was isolated among 10 candidate tolerance mutants. Inactivation of psaA was previously shown to result in reduced virulence of S. pneumoniae (as judged by intranasal or intraperitoneal challenge of mice) and in reduced adherence to A549 cells (type II pneumocytes), leading to the suggestion that PsaA was an adhesin (Berry and Paton, 1996, Infect Immun64: 5255–5262). This gene is part of the psa locus (Fig. 1) that encodes an ATP-binding cassette (ABC) permease belonging to cluster 9, a family of ABC metal permeases (Dintilhac et al., 1997, Mol Microbiol25: 727–740). . Organization of the psa locus. ORFs are indicated by shaded arrows; psaB, psaC, psaA and psaD encode an ATP-binding cassette (ABC) protein, a hydrophobic membrane protein, a Mn-binding lipoprotein and a putative thiol peroxidase respectively. Arrows above and below the map define the limits of IDM-targeting fragments used for disruption of the psaA or the psaD (ORF3) genes, respectively, by: N, Novak et al. (1998, Mol Microbiol 29: 1285–1296); B, Berry and Paton (1996, Infect Immun 64: 5255–5262); D, Dintilhac et al. (1997, Mol Microbiol 25: 727–740). The 3.1 kb and the less abundant 0.6 kb transcripts detected with a psaD probe (Novak et al. 1998, Mol Microbiol 29: 1285–1296) are also indicated. It has been suggested that transcription of the psa locus is initiated from P1, a putative promoter located upstream of psaB. A second weaker promoter, P2 located immediately upstream of psaD, was postulated by Novak et al. (1998, Mol Microbiol 29: 1285–1296) to account for the shortest transcript. However, the latter could as well result from the processing of the 3.1 kb transcript. Taking into account the respective amount of each transcript and depending on the existence of P2, the expression of psaD could be predicted to be severely reduced, or totally abolished, in strains with polar plasmid insertions in psaA (or further upstream in the operon). Novak et al. (1998, Mol Microbiol29: 1285–1296) reported that psa mutants displayed pleiotropic phenotypes: (i) reduced sensitivity to the lytic and killing effects of penicillin; (ii) growth in chains of 40–50 (psaC ) to 200–300 (psaD ) cells; (iii) autolysis defect and loss of sensitivity to low concentrations of deoxycholate (DOC), a species characteristic trait; (iv) absence of LytA, the major autolytic amidase; (v) almost complete loss of choline-binding proteins (ChBPs) (psaC and psaD ) and absence of CbpA; (vi) loss of transformability (except psaA); and (vii) manganese (Mn) requirement for growth in a chemically defined medium. Because penicillin tolerance was first associated with an autolysis defect (Tomasz et al., 1970, Nature227: 138–140), the absence of LytA (phenotype iv) could itself explain phenotypes i and iii. Dysregulation of lytA could not be investigated because, according to Novak et al. (1998, Mol Microbiol29: 1285–1296), the difficulty in lysing psa mutant cells prohibited Northern analysis, although lysates of the psa mutants could be obtained for immunoblot analysis of LytA and of RecA and for Southern confirmation of the psa mutations. Nevertheless, because expression of the lytA gene has been shown to be driven by three different promoters, including Pb which is the recA basal promoter (Mortier-Barrière et al., 1998, Mol Microbiol27: 159–170), and because wild-type levels of RecA were detected in the psa mutants (Novak et al., 1998, Mol Microbiol29: 1285–1296), it seems difficult to account for the complete absence of LytA on the basis of altered expression. On the other hand, phenotypes i–iv are reminiscent of alterations observed after the replacement of choline (Ch) by ethanolamine (EA) in the cell wall of pneumococcus (Tomasz, 1968, Proc Natl Acad Sci USA59: 86–93). Similar phenotypes were also displayed by Ch-independent mutants of S. pneumoniae (Severin et al., 1997, Microb Drug Res3: 391–400; Yother et al., 1998, J Bacteriol180: 2093–2101). S. pneumoniae has a nutritional requirement for Ch that is incorporated by covalent bonds into the cell wall teichoic acids (TA) and in the membrane-bound lipoteichoic acid (LTA). Ch residues bound to TA (ChTA) were shown to be absolutely required for LytA activity (Holtje and Tomasz, 1975; J Biol Chem250: 6072–6076). The action of LytA has long been thought to be restricted to pneumococcal cell walls because of this requirement. However, recent reports suggest that ChTA is required only to relieve inhibition of LytA by TA (Díaz et al., 1996, Mol Microbiol19: 667–681; Severin et al., 1997, Microb Drug Res3: 391–400). Ch residues are also essential for surface attachment of a number of ChBPs (Garcia et al., 1999, Microb Drug Res4: 25–36), including PspA (Yother and White, 1994, J Bacteriol176: 2976–2985) and most probably LytB. The latter protein is a newly described murein hydrolase that is essential for cell separation (Garcia et al., 1999, Mol Microbiol, in press). An attractive hypothesis would then have been that the production of cell wall Ch was affected in the mutants studied by Novak and co-workers. However, this hypothesis was ruled out by immunoblotting with an antibody specific for phosphorylcholine (Novak et al., 1998, Mol Microbiol29: 1285–1296). In addition, such a hypothesis cannot explain the failure to detect LytA because this protein was not released from the cell in a Ch-independent mutant or when EA was substituted for Ch. This is in contrast to PspA, which requires the presence of Ch residues in the LTA for surface attachment (Yother and White, 1994, J Bacteriol176: 2976–2985; Yother et al., 1998, J Bacteriol180: 2093–2101). An alternative hypothesis that deserves further examination is that TA and/or LTA metabolism is affected in these mutants in such a way as to interfere with the attachment of LytA and of the other ChBPs to the pneumococcal cell surface. Although the Mn requirement for growth of psa mutants confirmed our findings (Dintilhac et al., 1997, Mol Microbiol25: 727–740), we felt concerned by phenotypes (iii) and (iv) because during our investigations we did not notice any autolysis defect in a psaA mutant. Therefore, the kinetics of DOC-triggered autolysis of our psaA mutant strain were reinvestigated. They appeared indistinguishable from that of the isogenic wild-type parent (Fig. 2, left). Western blot analysis was then performed using antibody to the LytA protein. Similar amounts of LytA were detected in the psaA mutant and in the parent strain (data not shown, but see below). Because the genetic background of our mutant differed slightly from that of the psa mutants of Novak and co-workers (CP compared with R6 parental strain), we transferred the psaA mutation to the latter strain. Normal DOC-triggered autolysis was also observed in the R6 mutant derivative (data not shown). . Kinetics of DOC-triggered autolysis (left) and Western blot analysis of LytA (right) in the psaA mutant and in the parent strain (wt ). Left. DOC was added (arrow) to psaA mutant (▪) and wt (▴) cultures in the exponential phase of growth in C medium (A). Comparison of the kinetics on an expanded time scale is shown in B. DOC concentration: 0.02% (filled symbols) or 0.05% (open symbols). Right. Western blot analysis of lysates of D39 and derivatives subjected to SDS–PAGE (12%) and probed using polyclonal mouse anti-LytA. Lanes (from left to right): 1, prestained molecular size markers (band sizes are 241, 147, 99, 69, 57, 43, 29, 23 and 18 kDa from top to bottom); 2, D39; 3, PsaA−; 4, PsaA+; 5, ORF3; 6, LytA−; 7, purified LytA; 8, molecular size markers. Susceptibility to autolysis, chain length and penicillin tolerance was also examined in derivatives of the S. pneumoniae type 2 strain D39 carrying insertion–duplication mutations (IDM) at three places in the psa operon. Strains PsaA− and ORF3 contained mutations in psaA and psaD (Fig. 1), respectively, whereas in strain PsaA+ the mutagenesis vector was inserted between the psaA and psaD open reading frames (Berry and Paton, 1996, Infect Immun64: 5255–5262). There was no difference in the rate of autolysis in the presence of 0.05% DOC between the wild-type D39 and any of the three mutants. Moreover, there was no apparent difference in the mean chain length in stationary-phase cultures of the four strains (≈6–8 cells in each case; results not shown). Lysates of D39 and the three mutants were also examined by Western blot using polyclonal anti-LytA. The intensity of the 36 kDa immunoreactive band that co-migrates with purified LytA was similar for the D39, PsaA−, PsaA+ and ORF3 lysates, but absent in the lysate of a derivative of D39 with an insertion–duplication mutation in lytA (Fig. 2, right). Finally, we tested D39, PsaA−, PsaA+ and ORF3 for penicillin tolerance. At a dose of 0.2 μg ml−1 penicillin G (10 times the MIC of D39) over a 6 h period, there was no difference in the rate of penicillin-induced lysis or killing, as judged by decrease in absorbance at 600 nm and viable count, respectively, between any of the four strains (result not shown). There was also no difference in the rate of penicillin-induced lysis in D39 cultures grown in the presence or absence of a 1:100 dilution of polyclonal anti-PsaA. Collectively, all these observations suggest that the use of PsaA in a pneumococcal vaccine formulation, which was questioned on the basis of the possible promotion of penicillin tolerance (Novak et al., 1998, Mol Microbiol29: 1285–1296), should still be considered. How can we account for the conflicting observations? All psaA mutants were generated by IDM, but using different psaA-targeting fragments (Fig. 1). Although the truncated psaA gene in the CP and D39 mutants is about 260 nucleotides longer than in the mutant of Novak and co-workers, the CP mutation resulted in complete loss of the PsaA protein, as observed by Western blot analysis (Dintilhac et al., 1997, Mol Microbiol25: 727–740). Different non-replicative plasmids were used to construct the psaA mutants, pBluescript (Dintilhac et al., 1997, Mol Microbiol25: 727–740), pVA891 (Berry and Paton, 1996, Infect Immun64: 5255–5262) or pJDC9 (Novak et al., 1998, Mol Microbiol29: 1285–1296). Interestingly, unlike the other two plasmids, pJDC9 contains strong transcriptional terminators (Chen and Morrison, 1987, Gene55: 179–187). It is, therefore, possible that insertion of pJDC9 to generate the psaBCA mutants resulted in stronger polar effects on psaD expression than in the other mutants (see legend to Fig. 1). However, ‘silencing’ of psaD is unlikely to be the explanation for the pleiotropic phenotype of the various psa mutants of Novak and co-workers because the phenotype of the D39 psaD (ORF3) mutant was indistinguishable from D39. At the moment, no explanation(s) that could satisfactorily account for the discrepancy can be proposed. Nevertheless, as stated above, induction of pleiotropic effects including autolysis defects and penicillin tolerance is clearly not a universal feature of psa mutants, and therefore PsaA remains a potential pneumococcal vaccine target worthy of careful consideration.