Prediction of resistance to erythromycin in the genus Rickettsia by mutations in L22 ribosomal protein

J. M. Rolain and D. Raoult*

Unité desxs Rickettsies, CNRS UMR 6020, IFR 48, Faculté de Médecine, Université de la Méditerranée, 27 Bd Jean Moulin, 13385 Marseille Cedex 05, France


* Corresponding author. Tel: +33-4-91-38-55-17; Fax: +33-4-91-83-03-90; E-mail: Didier.Raoult{at}medecine.univ-mrs.fr

Received 6 January 2005; returned 19 May 2005; revised 7 May 2005; accepted 15 June 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Typhus group (TG) rickettsiae are naturally susceptible to erythromycin whereas spotted fever group (SFG) rickettsioses are not. The aim of this study was to compare in silico genetic determinants known to be associated with resistance to macrolide compounds.

Methods and results: Available sequences of the 23S RNA gene, and L4 and L22 ribosomal proteins of rickettsial strains were aligned and compared using in silico methods. Although there were no sequence differences in domain V of the 23S RNA gene and in the conserved region of the L4 ribosomal protein gene, we found that TG rickettsiae had a triple amino acid difference in the highly conserved region of the L22 ribosomal protein compared with the SFG rickettsiae.

Conclusions: We believe that the triple amino acid difference in the L22 ribosomal protein found in this study may explain the difference in susceptibility to erythromycin among the Rickettsia genus. Genome analysis may help to predict possible molecular mechanisms of resistance for fastidious and intracellular bacteria and cloning and expression of such proteins should be investigated in the future in order to prove our hypothesis.

Keywords: antibiotic resistance , genome analysis , in silico , L22 protein , erythromycin resistance , macrolides


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Rickettsiae are strict intracellular bacteria belonging to the alpha group of Proteobacteria. Rickettsioses are zoonoses that are geographically distributed according to the distribution of their infected vectors. The genus comprises typhus group (TG) rickettsiae which includes Rickettsia prowazekii, the agent of epidemic typhus, and Rickettsia typhi, the agent of murine typhus; and spotted fever group (SFG) rickettsiae. There are at least four main phenotypic differences between these two subgroups. Optimal temperature for growth in cell culture is 32°C for the SFG rickettsiae, and 35°C for the TG rickettsiae. SFG rickettsiae can be observed in the nuclei of host cells whilst TG rickettsiae are observed exclusively in the cytoplasm.1 The cytopathic effects of the SFG rickettsiae are rapid and important, leading to the formation of large plaques in culture, whereas the cytopathic effects of the TG rickettsiae are less important and rapid.2 Finally, the typhus group is susceptible to erythromycin (MICs from 0.25 to 1 mg/L) whereas the spotted fever group is not (MICs from 1 to 8 mg/L).2 These phenotypic differences have been partially elucidated from comparison of the genome of R. prowazekii and Rickettsia conorii.1 However, the natural mechanism of resistance of SFG rickettsia to erythromycin is not known.

Macrolide compounds inhibit protein synthesis by binding to domains II and V of 23S rRNA.3 The first mechanism of macrolide resistance described was due to post-transcriptional modifications of the 23S rRNA by the adenine-N6 methyltransferase encoded by erm genes.4 Two other mechanisms of resistance to macrolides have been described: target site modification, and active efflux of the antibiotic from the cell.4 Antibiotic target-site modification by mutations in domain V of 23S rRNA could also confer resistance to several MLSB antibiotics.4 In certain bacterial species, macrolide resistance can be due to mutations in the highly conserved region of ribosomal proteins L4 and L22.5 A number of different antibiotic resistance genes code for efflux proteins, which pump the antibiotic out of the cell or the cellular membrane, keeping intracellular concentrations low and ribosomes free from antibiotic. Many of these proteins [mef(A), mef(E)] have homology to the major facilitator superfamily (MFS), the resistance/nodulation/division (RND) superfamily (AcrAB-TolC, mex, mtrD, amrB), or the ATP binding cassette (ABC) transporter [msr(A), lmr(A)].4

In this study, we have investigated the resistance mechanisms of bacteria of the genus Rickettsia to erythromycin by in silico comparison for the presence of known macrolide resistance determinants and by sequence comparison of genes encoding 23S rRNA and ribosomal proteins L4 and L22.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antibiotic resistance determinants known to be target genes involved in molecular resistance to macrolide compounds were retrieved from available genomes at the KEGG Encyclopedia web site (http://www.genome.jp/kegg/kegg2.html). Sequences of the 23S rRNA gene and ribosomal proteins L4 and L22 were retrieved at the KEGG Encyclopedia web site (http://www.genome.jp/kegg/kegg2.html) for R. conorii strain Malish 7, R. typhi strain Wilmington, and R. prowazekii strain Madrid E or at the NCBI web site (http://www.ncbi.nlm.nih.gov/) for Rickettsia sibirica 246 (GenBank accession number NZ_AABW00000000), Rickettsia akari strain Hartford (GenBank accession number NZ_AAFE00000000) and Rickettsia rickettsii (GenBank accession number NZ_AADJ00000000). For three species (Rickettsia africae, Rickettsia belli and Rickettsia felis), the corresponding sequences were retrieved from ongoing genome sequences available in our laboratory (unpublished data). The nucleotide sequence of the 23S rRNA gene and amino acid sequences of the ribosomal proteins L4 and L22 were compared and aligned to look at possible mutations known to be associated with macrolide resistance4 using the CLUSTALW program supported by the Infobiogen web site (www.infobiogen.fr). Sequence alignments were compared with known original sequences of Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli and Haemophilus influenzae retrieved at the KEGG Encyclopedia web site.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We did not find any erm gene in the available genome sequences of rickettsial strains. Sequences of the 23S rRNA of the nine rickettsial strains did not display any nucleotide difference in positions 754, 2057, 2058, 2059 and 2611 (E. coli numbering). Moreover, the two regions of domains II and V were identical among the nine rickettsial strains. Similarly, the highly conserved region of the L4 protein (63KPWRQKGTGRAR74, S. pneumoniae numbering) did not show any amino acid differences or insertions or deletions. Conversely, we found three amino acid differences between TG rickettsia and SFG rickettsia in the highly conserved region at the C terminus of the L22 protein (Figure 1). R. typhi and R. prowazekii had two amino acid changes at positions 83 and 84 and a single amino acid change at position 89 (S. pneumoniae numbering) compared with the seven SFG rickettsial strains. Finally, we found that the genome of R. conorii contains six encoding genes for the RND superfamily and 20 encoding genes similar to that of ABC transporters whereas R. prowazekii and R. typhi did not contain RND superfamily encoding genes and less encoding genes for ABC transporters (4 and 12, respectively).



View larger version (36K):
[in this window]
[in a new window]
 
Figure 1. Amino acid sequence of the C-terminus of the L22 protein (from amino acid 60 to 116) for rickettsial strains and reference strains of E. coli, S. aureus, H. influenzae and S. pneumoniae. Amino acid differences between SFG and TG rickettsiae are indicated in boldface and are underlined. Abbreviations: V, variable; R, resistant; S, susceptible.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Description of resistance determinants for intracellular bacteria remains a challenge since in vitro mutants are very difficult to obtain. The recent description of molecular mechanisms of resistance to erythromycin lead us to look at possible mutations within the drug target by genome comparisons. We have previously reported the same approach to explain the intrinsic resistance of Ehrlichia spp., Anaplasma phagocytophilum and Wolbachia pipientis to macrolide compounds due to nucleotide substitutions in domains II and V of the 23S rRNA gene.6 Similarly, we have reported the natural resistance to rifampicin in several species of Rickettsia due to mutations in the rpoB gene.7 Our preliminary results indicate that natural resistance to erythromycin in SFG rickettsia could be due to alterations in the C terminus of the ribosomal protein L22, a protein of 119 amino acids (Figure 1). Indeed, the three amino acid differences found between the two subgroups of rickettsiae are located in a highly conserved region of the L22 protein. In E. coli, a deletion of three amino acids in this conserved region (Met82-Lys83-Arg84) conferred resistance to erythromycin.5 Similarly, amino acid substitutions as well as insertions or deletions within the region between amino acid positions 80 and 94 have been reported in in vitro mutants of H. influenzae resistant to macrolide compounds.8 In S. aureus, deletions or insertions have been found in laboratory mutants resistant to macrolides, in particular the deletion of two amino acids at positions 79 and 80.9 Finally, single amino acid mutations (G95D, P99Q, K94Q) or triple amino acid substitutions (A93E-P91S-G83E) have been reported in macrolide-resistant mutants of S. pneumoniae.5 Finally, since the genome of R. conorii contains more efflux encoding genes compared with TG rickettsiae, this may also be involved in the difference in susceptibility to macrolide compounds between these two subgroups.

In conclusion, we hypothesize that amino acid differences found in the highly conserved region of the L22 ribosomal protein of rickettsial strains may explain the natural difference in susceptibility to erythromycin between TG and SFG rickettsia. Cloning and expression of such proteins or introduction of these amino acids by site-directed mutagenesis into a suitable susceptible host should be investigated in the future in order to prove our hypothesis. Alternatively, transformation systems could be used to mutate a predicted residue in sensitive rickettsiae, as was demonstrated for rifampicin resistance.10 Nevertheless, neither transformation nor spontaneous mutants of the typhus group rickettsiae for erythromycin resistance are easily obtained in vitro. In our experience, we have been unable to generate such mutants (personal observation). We believe that genome analysis may help to predict possible molecular mechanisms of resistance for fastidious and intracellular bacteria for which transformation assays are usually not suitable.


    Acknowledgements
 
We thank Esther Platt for reviewing the manuscript prior to submission.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1. Ogata H, Audic S, Renesto-Audiffren P et al. Mechanisms of evolution in Rickettsia conorii and R. prowazekii. Science 2001; 293: 2093–8.[Abstract/Free Full Text]

2. Rolain JM, Maurin M, Vestris G et al. In vitro susceptibilities of 27 rickettsiae to 13 antimicrobials. Antimicrob Agents Chemother 1998; 42: 1537–41.[Abstract/Free Full Text]

3. Schlunzen F, Harms JM, Franceschi F et al. Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature 2001; 413: 814–21.[CrossRef][ISI][Medline]

4. Vester B, Douthwaite S. Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob Agents Chemother 2001; 45: 1–12.[Free Full Text]

5. Canu A, Malbruny B, Coquemont M et al. Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae. Antimicrob Agents Chemother 2002; 46: 125–31.[Abstract/Free Full Text]

6. Branger S, Rolain JM, Raoult D. Evaluation of antibiotic susceptibilities of Ehrlichia canis, Ehrlichia chaffensis, and Anaplasma phagocytophilum by real-time PCR. Antimicrob Agents Chemother 2004; 48: 4822–8.[Abstract/Free Full Text]

7. Drancourt M, Raoult D. Characterization of mutations in the rpoB gene in naturally rifampin-resistant Rickettsia species. Antimicrob Agents Chemother 1999; 43: 2400–3.[Abstract/Free Full Text]

8. Kosowska K, Credito K, Pankuch GA et al. Activities of two novel macrolides, GW 773546 and GW 708408, compared with those of telithromycin, erythromycin, azithromycin, and clarithromycin against Haemophilus influenzae. Antimicrob Agents Chemother 2004; 48: 4113–9.[Abstract/Free Full Text]

9. Malbruny B, Canu A, Bozdogan B et al. Resistance to quinupristin–dalfopristin due to mutation of L22 ribosomal protein in Staphylococcus aureus. Antimicrob Agents Chemother 2002; 46: 2200–7.[Abstract/Free Full Text]

10. Rachek LI, Tucker AM, Winkler HH et al. Transformation of Rickettsia prowazekii to rifampin resistance. J Bacteriol 1998; 180: 2118–24.[Abstract/Free Full Text]