Vink, C.

Functional characterization of the RuvB homologs from Mycoplasma pneumoniae and Mycoplasma genitalium (Article)

2011-12-01T00:00:00Z

Homologous recombination between repeated DNA elements in the genomes of Mycoplasma species has been hypothesized to be a crucial causal factor in sequence variation of antigenic proteins at the bacterial surface. To investigate this notion, studies were initiated to identify and characterize the proteins that form part of the homologous DNA recombination machinery in Mycoplasma pneumoniae as well as Mycoplasma genitalium. Among the most likely participants of this machinery are homologs of the Holliday junction migration motor protein RuvB. In both M. pneumoniae and M. genitalium, genes have been identified that have the capacity to encode RuvB homologs (MPN536 and MG359, respectively). Here, the characteristics of the MPN536- and MG359-encoded proteins (the RuvB proteins from M. pneumoniae strain FH [RuvBFH] and M. genitalium [RuvBMge], respectively) are described. Both RuvBFHand RuvBMgewere found to have ATPase activity and to bind DNA. In addition, both proteins displayed divalent cation- and ATP-dependent DNA helicase activity on partially double-stranded DNA substrates. The helicase activity of RuvBMge, however, was significantly lower than that of RuvBFH. Interestingly, we found RuvBFHto be expressed exclusively by subtype 2 strains of M. pneumoniae. In strains belonging to the other major subtype (subtype 1), a version of the protein is expressed (the RuvB protein from M. pneumoniae strain M129 [RuvBM129]) that differs from RuvBFHin a single amino acid residue (at position 140). In contrast to RuvBFH, RuvBM129displayed only marginal levels of DNAunwinding activity. These results demonstrate that M. pneumoniae strains (as well as closely related Mycoplasma spp.) can differ significantly in the function of components of their DNA recombination and repair machinery.

Sequence variation within the P1 gene of Mycoplasma pneumoniae (Article)

2011-10-01T00:00:00Z

Identification of amino acid residues critical for catalysis of holliday junction resolution by Mycoplasma genitalium RecU (Article)

2011-08-01T00:00:00Z

The RecU protein from Mycoplasma genitalium, RecUMge, is a 19.4-kDa Holliday junction (HJ) resolvase that binds in a nonspecific fashion to HJ substrates and, in the presence of Mn2+, cleaves these substrates at a specific sequence (5'-G/TC2C/TTA/GG-3'). To identify amino acid residues that are crucial for HJ binding and/or cleavage, we generated a series of 16 deletion mutants (9 N- and 7 C-terminal deletion mutants) and 31 point mutants of RecUMge. The point mutations were introduced at amino acid positions that are highly conserved among bacterial RecU-like sequences. All mutants were purified and tested for the ability to bind to, and cleave, HJ substrates. We found the five N-terminal and three C-terminal amino acid residues of RecUMgeto be dispensable for its catalytic activities. Among the 31 point mutants, 7 mutants were found to be inactive in both HJ binding and cleavage. Interestingly, in 12 other mutants, these two activities were uncoupled; while these proteins displayed HJ-binding characteristics similar to those of wild-type RecUMge, they were unable to cleave HJ substrates. Thus, 12 amino acid residues were identified (E11, K31, D57, Y58, Y66, D68, E70, K72, T74, K76, Q88, and L92) that may play either a direct or indirect role in the catalysis of HJ resolution.

Variation in a surface-exposed region of the Mycoplasma pneumoniae P40 protein as a consequence of homologous DNA recombination between RepMP5 elements (Article)

2011-02-01T00:00:00Z

Mycoplasma pneumoniae is a human pathogen that causes a range of respiratory tract infections. The first step in infection is adherence of the bacteria to the respiratory epithelium. This step is mediated by a specialized organelle, which contains several proteins (cytadhesins) that have an important function in adherence. Two of these cytadhesins, P40 and P90, represent the proteolytic products from a single 130 kDa protein precursor, which is encoded by the MPN142 gene. Interestingly, MPN142 contains a repetitive DNA element, termed RepMP5, of which homologues are found at seven other loci within the M. pneumoniae genome. It has been hypothesized that these RepMP5 elements, which are similar but not identical in sequence, recombine with their counterpart within MPN142 and thereby provide a source of sequence variation for this gene. As this variation may give rise to amino acid changes within P40 and P90, the recombination between RepMP5 elements may constitute the basis of antigenic variation and, possibly, immune evasion by M. pneumoniae. To investigate the sequence variation of MPN142 in relation to inter-RepMP5 recombination, we determined the sequences of all RepMP5 elements in a collection of 25 strains. The results indicate that: (i) inter-RepMP5 recombination events have occurred in seven of the strains, and (ii) putative RepMP5 recombination events involving MPN142 have induced amino acid changes in a surface-exposed part of the P40 protein in two of the strains. We conclude that recombination between RepMP5 elements is a common phenomenon that may lead to sequence variation of MPN142-encoded proteins.

Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing (Article)

2010-09-01T00:00:00Z

The first choice antibiotics for treatment of Mycoplasma pneumoniae infections are macrolides. Several recent studies, however, have indicated that the prevalence of macrolide (ML)-resistance, which is determined by mutations in the bacterial 23S rRNA, is increasing among M. pneumoniae isolates. Consequently, it is imperative that ML-resistance in M. pneumoniae is rapidly detected to allow appropriate and timely treatment of patients. We therefore set out to determine the utility of pyrosequencing as a convenient technique to assess ML-resistance. In addition, we studied whether pyrosequencing could be useful for molecular typing of M. pneumoniae isolates. To this end, a total of four separate pyrosequencing assays were developed. These assays were designed such as to determine a short genomic sequence from four different sites, i.e. two locations within the 23S rRNA gene, one within the MPN141 (or P1) gene and one within the MPN528a gene. While the 23S rRNA regions were employed to determine ML-resistance, the latter two were used for molecular typing. The pyrosequencing assays were performed on a collection of 108 M. pneumoniae isolates. The ML-resistant isolates within the collection (n = 4) were readily identified by pyrosequencing. Moreover, each strain was correctly typed as either a subtype 1 or subtype 2 strain by both the MPN141 and MPN528a pyrosequencing test. Interestingly, two recent isolates from our collection, which were identified as subtype 2 strains by the pyrosequencing assays, were found to carry novel variants of the MPN141 gene, having rearrangements in each of the two repetitive elements (RepMP4 and RepMP2/3) within the gene. In conclusion, pyrosequencing is a convenient technique for ML-resistance determination as well as molecular typing of M. pneumoniae isolates.

The Mycoplasma genitalium MG352-encoded protein is a Holliday junction resolvase that has a non-functional orthologue in Mycoplasma pneumoniae (Article)

2010-09-01T00:00:00Z

Recombination between repeated DNA elements in the genomes of Mycoplasma species appears to lie at the basis of antigenic variation of several essential surface proteins. It is therefore imperative that the DNA recombinatorial pathways in mycoplasmas be unravelled. Here, we describe the proteins encoded by the Mycoplasma genitalium MG352 and Mycoplasma pneumoniae MPN528a genes (RecUMgeand RecUMpnrespectively), which share sequence similarity with RecU Holliday junction (HJ) resolvases. RecUMgewas found to: (i) bind HJ substrates and large double-stranded DNA molecules and (ii) cleave HJ substrates at the sequence 5′-G/TC↓C/TTA/GG- 3′ in the presence of Mn2+. Interestingly, RecUMpn(from M. pneumoniae subtype 2 strains) did not possess obvious DNA binding or cleavage activities, which was found to be caused by the presence of a glutamic acid residue at position 67 of the protein, which is not conserved in RecUMge. Additionally, RecUMpnappears not to be expressed by subtype 1 M. pneumoniae strains, as these possess a TAA translation termination codon at position 181-183 of MPN528a. We conclude that RecUMgeis a HJ resolvase that may play a central role in recombination in M. genitalium.

Identification and classification of P1 variants of Mycoplasma pneumoniae (Article)

2010-02-01T00:00:00Z

The Mycoplasma pneumoniae MPN490 and Mycoplasma genitalium MG339 genes encode RecA homologs that promote homologous DNA strand exchange (Article)

2009-11-01T00:00:00Z

The P1, P40, and P90 proteins of Mycoplasma pneumoniae and the MgPa and P110 proteins of Mycoplasma genitalium are immunogenic adhesion proteins that display sequence variation. Consequently, these proteins are thought to play eminent roles in immune evasive strategies. For each of the five proteins, a similar underlying molecular mechanism for sequence variation was hypothesized, i.e., modification of the DNA sequences of their respective genes. This modification is thought to result from homologous recombination of parts of these genes with repeat elements (RepMp and MgPar elements in M. pneumoniae and M. genitalium, respectively) that are dispersed throughout the bacterial genome. Proteins that are potentially involved in homologous DNA recombination have been suggested to be implicated in recombination between these repeat elements and thereby in antigenic variation. To investigate this notion, we set out to study the function of the RecA homologs that are encoded by the M. pneumoniae MPN490 and M. genitalium MG339 genes. Both proteins, which are 79% identical on the amino acid level, were found to promote recombination between homologous DNA substrates in an ATP-dependent fashion. The recombinational activities of both proteins were Mg2+and pH dependent and were strongly supported by the presence of single-stranded DNA binding protein, either from M. pneumoniae or from Escherichia coli. We conclude that the MPN490- and MG339-encoded proteins are RecA homologs that have the capacity to recombine homologous DNA substrates. Thus, they may play a central role in recombination between repetitive elements in both M. pneumoniae and M. genitalium. Copyright

Sequence variations in RepMP2/3 and RepMP4 elements reveal intragenomic homologous DNA recombination events in Mycoplasma pneumoniae (Article)

2009-09-14T00:00:00Z

The gene encoding major adhesin protein P1 of Mycoplasma pneumoniae, MPN141, contains two DNA sequence stretches, designated RepMP2/3 and RepMP4, which display variation among strains. This variation allows strains to be differentiated into two major P1 genotypes (1 and 2) and several variants. Interestingly, multiple versions of the RepMP2/3 and RepMP4 elements exist at other sites within the bacterial genome. Because these versions are closely related in sequence, but not identical, it has been hypothesized that they have the capacity to recombine with their counterparts within MPN141, and thereby serve as a source of sequence variation of the P1 protein. In order to determine the variation within the RepMP2/3 and RepMP4 elements, both within the bacterial genome and among strains, we analysed the DNA sequences of all RepMP2/3 and RepMP4 elements within the genomes of 23 M. pneumoniae strains. Our data demonstrate that: (i) recombination is likely to have occurred between two RepMP2/3 elements in four of the strains, and (ii) all previously described P1 genotypes can be explained by inter-RepMP recombination events. Moreover, the difference between the two major P1 genotypes was reflected in all RepMP elements, such that subtype 1 and 2 strains can be differentiated on the basis of sequence variation in each RepMP element. This implies that subtype 1 and subtype 2 strains represent evolutionarily diverged strain lineages. Finally, a classification scheme is proposed in which the P1 genotype of M. pneumoniae isolates can be described in a sequence-based, universal fashion.

Raman spectroscopic typing reveals the presence of carotenoids in Mycoplasma pneumoniae (Article)

2009-08-10T00:00:00Z

Raman spectroscopy has previously been demonstrated to be a highly useful methodology for the identification and/or typing of micro-organisms. In this study, we set out to evaluate whether this technology could also be applied as a tool to discriminate between isolates of Mycoplasma pneumoniae, which is generally considered to be a genetically highly uniform species. In this evaluation, a total of 104 strains of M. pneumoniae were analysed, including two reference strains (strains M129 and FH), and 102 clinical isolates, which were isolated between 1973 and 2005 and originated from various countries. By Raman spectral analysis (Raman typing) of this strain collection, we were able to reproducibly distinguish six different clusters of strains. An unequivocal correlation between Raman typing and P1 genotyping, which is based on sequence differences in the P1 (or MPN141) gene of M. pneumoniae, was not observed. In the two major Raman clusters that we identified (clusters 3 and 6, which together harboured 81% of the strains), the different P1 subtypes were similarly distributed, and ∼76% isolates were of subtype 1, ∼20% of subtype 2 and ∼5% of variant 2a. Nevertheless, a relatively high prevalence of P1 subtype 2 strains was found in clusters 2 and 5 (100 %), as well as in cluster 1 (75 %) and cluster 4 (71 %); these clusters, however, harboured a small number of strains. Only two of the strains (2 %) could not be typed correctly. Interestingly, analysis of the Raman spectra revealed the presence of carotenoids in M. pneumoniae. This finding is in line with the identification of M. pneumoniae genes that have similarity with genes involved in a biochemical pathway leading to carotenoid synthesis, i.e. the 2-C -methyl-D-erythritol 4-phosphate (MEP) pathway. Therefore, we hypothesize that M. pneumoniae hosts an MEP-like pathway for carotenoid synthesis. We conclude that Raman spectroscopy is a convenient tool for discriminating between M. pneumoniae strains, and that it presents a promising supplement to the current methods for typing of this bacterium.

Evaluation of MeningoFinder, a novel multiplex ligation-dependent probe amplification assay for simultaneous detection of six virus species causing central nervous system infections (Article)

2009-08-01T00:00:00Z

A multiplex ligation-dependent probe amplification assay for simultaneous detection of six virus species was developed and tested on clinical cerebrospinal fluid (CSF) samples. The assay, termed MeningoFinder, showed an accordance of 97%, concordance of 96%, interlaboratory sensitivity of 90%, and interlaboratory specificity of 94% compared to PCRs. Copyright

Anti-staphylococcal humoral immune response in persistent nasal carriers and noncarriers of Staphylococcus aureus (Article)

2009-03-01T00:00:00Z

BACKGROUND. Persistent carriers have a higher risk of Staphylococcus aureus infections than noncarriers but a lower risk of bacteremia-related death. Here, the role played by anti-staphylococcal antibodies was studied. METHODS. Serum samples from 15 persistent carriers and 19 noncarriers were analyzed for immunoglobulin (Ig) G, IgA, and IgM binding to 19 S. aureus antigens, by means of Luminex technology. Nasal secretions and serum samples obtained after 6 months were also analyzed. RESULTS. Median serum IgG levels were significantly higher in persistent carriers than in noncarriers for toxic shock syndrome toxin (TSST)-1 (median fluorescence intensity [MFI] value, 11,554 vs. 4291; P < .001) and staphylococcal enterotoxin (SE) A (742 vs. 218; P < .05); median IgA levels were higher for TSST-1 (P < .01), SEA, and clumping factor (Clf) A and B (P < .05). The in vitro neutralizing capacity of anti-TSST-1 antibodies was correlated with the MFI value (R(2) = 0.93) and was higher in persistent carriers (90.6% vs. 70.6%; P < .05). Antibody levels were stable over time and correlated with levels in nasal secretions (for IgG, R(2) = 0.87; for IgA, R(2) = 0.77). CONCLUSIONS. Antibodies to TSST-1 have a neutralizing capacity, and median levels of antibodies to TSST-1, SEA, ClfA, and ClfB are higher in persistent carriers than in noncarriers. These antibodies might be associated with the differences in the risk and outcome of S. aureus infections between nasal carriers and noncarriers.

Chemokines and chemokine receptors encoded by cytomegaloviruses (Article)

2008-12-01T00:00:00Z

CMVs carry several genes that are homologous to genes of the host organism. These include genes homologous to those encoding chemokines (CKs) and G protein-coupled receptors (GPCRs). It is generally assumed that these CMV genes were hijacked from the host genome during the long co-evolution of virus and host. In light of the important function of the CK and GPCR families in the normal physiology of the host, it has previously been hypothesized that the CMV homologs of these proteins, CMV vCKs and vGPCRs, may also have a significant impact on this physiology, such that lifelong maintenance and/or replication of the virus within the infected host is guaranteed. In addition, several of these homologs were reported to have a major impact in the pathogenesis of infection. In this review, the current state of knowledge on the CMV vCKs and vGPCRs will be discussed.

The Mycoplasma pneumoniae MPN229 gene encodes a protein that selectively binds single-stranded DNA and stimulates Recombinase A-mediated DNA strand exchange (Article)

2008-10-31T00:00:00Z

Background. Mycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. In spite of this genetic stability, homologous DNA recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, P1, of M. pneumoniae. In order to identify the proteins that may be involved in homologous DNA recombination in M. pneumoniae, we set out to characterize the MPN229 open reading frame (ORF), which bears sequence similarity to the gene encoding the single-stranded DNA-binding (SSB) protein of other micro-organisms. Results. The MPN229 ORF has the capacity to encode a 166-amino acid protein with a calculated molecular mass of 18.4 kDa. The amino acid sequence of this protein (Mpn SSB) is most closely related to that of the protein predicted to be encoded by the MG091 gene from Mycoplasma genitalium (61% identity). The MPN229 ORF was cloned, and different versions of Mpn SSB were expressed in E. coli and purified to > 95% homogeneity. The purified protein was found to exist primarily as a homo-tetramer in solution, and to strongly and selectively bind single-stranded DNA (ssDNA) in a divalent cation- and DNA substrate sequence-independent manner. Mpn SSB was found to bind with a higher affinity to ssDNA substrates larger than 20 nucleotides than to smaller substrates. In addition, the protein strongly stimulated E. coli Recombinase A (RecA)-promoted DNA strand exchange, which indicated that Mpn SSB may play an important role in DNA recombination processes in M. pneumoniae. Conclusion. The M. pneumoniae MPN229 gene encodes a protein, Mpn SSB, which selectively and efficiently binds ssDNA, and stimulates E. coli RecA-promoted homologous DNA recombination. Consequently, the Mpn SSB protein may play a crucial role in DNA recombinatorial pathways in M. pneumoniae. The results from this study will pave the way for unraveling these pathways and assess their role in antigenic variation of M. pneumoniae.

Generation of polyclonal antibodies directed against G protein-coupled receptors using electroporation-aided DNA immunization (Article)

2008-07-01T00:00:00Z

Introduction: The generation of antibodies against G protein-coupled receptors (GPCRs) can be technically challenging. A modified DNA immunization protocol was employed in order to generate polyclonal antibodies against two herpes virus-encoded GPCRs, i.e. Epstein-Barr virus (EBV) pBILF1 and rat cytomegalovirus (RCMV) pR78. Methods: pBILF1 and pR78 expression plasmids were first injected into the tibialis anterior muscle of rats and rabbits, respectively. Subsequently, the uptake of plasmids by the muscle cells was facilitated through in vivo electroporation. Results: Potent antisera against both vGPCRs were obtained, as determined by immunoblot analysis and immunofluorescence. By using the antisera, we were able to show that the EBV BILF1 protein is expressed as a 45-kD, glycosylated protein, and that it is localized in the cytoplasmic membrane of EBV-infected cells. Interestingly, we found the R78-encoded vGPCRs to have unusual perinuclear localization in both R78-transfected and RCMV-infected cells. Discussion: The in vivo DNA electroporation method is a useful technique for generating antibodies against GPCRs.

Multicenter comparison of molecular methods for detection of Legionella spp. in sputum samples (Article)

2007-10-01T00:00:00Z

Legionellosis can be diagnosed by PCR using sputum samples. In this report, the methods of nine laboratories for 12 sputum samples with Legionella pneumophila and Legionella longbeachae are compared. We conclude that (i) liquefaction prevents PCR inhibition, (ii) the employed mip gene PCRs detected L. pneumophila only, and (iii) the 16S rRNA gene PCR detected both Legionella species and is preferred for the diagnosis of legionellosis. Copyright

Complementation between HIV integrase proteins mutated in different domains (Article)

1993-01-01T00:00:00Z

HIV integrase (IN) cleaves two nucleotides off the 3' end of viral DNA and integrates viral DNA into target DNA. Previously, three functional domains in the HIV IN protein have been identified: (i) the central catalytic domain, (ii) the C-terminal DNA binding domain, and (iii) the N-terminal region, which is also necessary for activity. We have now investigated whether IN proteins mutated in different domains can complement each other. Mutant D116I does not contain an intact active site, but does bind DNA, whereas the C-terminal deletion mutant C delta 73 does not bind DNA, but does have an intact active site. Neither mutant protein mediates site-specific cleavage or integration. However, a mixture of both proteins is active, suggesting that IN functions as an oligomer, and that two subunits can have different functions; one subunit binds the (viral) DNA and another subunit provides the active site. We found three classes of mutants, corresponding to the three domains mentioned above. Mutants from different classes, but not from the same class, can complement each other. However, complementation is most efficient when the N- and C-termini are present on the same molecule.

DNA binding properties of the integrase proteins of human immunodeficiency viruses types 1 and 2 (Article)

1991-01-01T00:00:00Z

Integration of retroviral DNA into the host chromosome requires the integrase protein (IN). We overexpressed the IN proteins of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) in E. coli and purified them. Both proteins were found to specifically cut two nucleotides off the ends of linear viral DNA, and to integrate viral DNA into target DNA. This demonstrates that HIV IN is the only protein required for integration of HIV DNA. Although the two types of IN proteins have only 53% amino acid sequence similarity, they act with equal efficiency on both type 1 and type 2 viral DNA. Binding of IN to DNA was tested: purified IN does not bind very specifically to viral DNA ends. Nevertheless, only viral DNA ends are cleaved and integrated. We interpret this as follows: in vitro quick aspecific binding to DNA is followed by slow specific cutting and integration. IN can not find viral DNA ends in the presence of an excess of aspecific DNA; in vivo this is not required since the IN protein is in constant proximity of viral DNA in the viral core particle.

Human immunodeficiency virus integrase protein requires a subterminal position of its viral DNA recognition sequence for efficient cleavage (Article)

1991-01-01T00:00:00Z

Retroviral integration requires cis-acting sequences at the termini of linear double-stranded viral DNA and a product of the retroviral pol gene, the integrase protein (IN). IN is required and sufficient for generation of recessed 3' termini of the viral DNA (the first step in proviral integration) and for integration of the recessed DNA species in vitro. Human immunodeficiency virus type 1 (HIV-1) IN, expressed in Escherichia coli, was purified to near homogeneity. The substrate sequence requirements for specific cleavage and integration of retroviral DNA were studied in a physical assay, using purified IN and short duplex oligonucleotides that correspond to the termini of HIV DNA. A few point mutations around the IN cleavage site substantially reduced cleavage; most other mutations did not have a drastic effect, suggesting that the sequence requirements are limited. The terminal 15 bp of the retroviral DNA were demonstrated to be sufficient for recognition by IN. Efficient specific cutting of the retroviral DNA by IN required that the cleavage site, the phosphodiester bond at the 3' side of a conserved CA-3' dinucleotide, be located two nucleotides away from the end of the viral DNA; however, low-efficiency cutting was observed when the cleavage site was located one, three, four, or five nucleotides away from the terminus of the double-stranded viral DNA. Increased cleavage by IN was detected when the nucleotides 3' of the CA-3' dinucleotide were present as single-stranded DNA. IN was found to have a strong preference for promoting integration into double-stranded rather than single-stranded DNA.

Integration of human immunodeficiency virus types 1 and 2 DNA in vitro by cytoplasmic extracts of Moloney murine leukemia virus-infected mouse NIH 3T3 cells (Article)

1990-01-01T00:00:00Z

An essential step in the life cycle of the human immunodeficiency virus (HIV) is integration of a DNA copy of the viral RNA into the genome of the infected cell. We show here that this step can be faithfully accomplished in vitro by the enzymatic machinery of another retrovirus, Moloney murine leukemia virus (MoMLV). Mini-HIV substrates, which are linearized plasmids with long terminal repeat sequences at their ends, were incubated with cytoplasmic extracts of MoMLV-infected NIH 3T3 cells and target DNA. The MoMLV integration apparatus carried out integration of the mini-HIV substrates correctly; the terminal nucleotides of the viral substrate were removed, and a 4-base-pair duplication of the target DNA flanked the inserted viral DNA (C. Shoemaker, S. P. Goff, E. Gilboa, M. Paskind, S. W. Mitra, and D. Baltimore, Proc. Natl. Acad. Sci. USA 77:3932-3936, 1980). Our experiments show that the substrate sequence requirements for integration in vitro were limited to a few nucleotides, as the similarity between HIV and MoMLV long terminal repeat ends is minimal.