RESUMEN
BACKGROUND: Mycoplasma pneumoniae is the most common bacterial cause of pneumonia in children hospitalised for community-acquired pneumonia (CAP). Prevention of infection by vaccines may be an important strategy in the presence of emerging macrolide-resistant M. pneumoniae. However, knowledge of immune responses to M. pneumoniae is limited, complicating vaccine design. METHODS: We studied the antibody response during M. pneumoniae respiratory tract infection and asymptomatic carriage in two different cohorts. RESULTS: In a nested case-control study (n=80) of M. pneumoniae carriers and matched controls we observed that carriage by M. pneumoniae does not lead to a rise in either mucosal or systemic M. pneumoniae-specific antibodies, even after months of persistent carriage. We replicated this finding in a second cohort (n=69) and also found that during M. pneumoniae CAP, mucosal levels of M. pneumoniae-specific IgA and IgG did increase significantly. In vitro adhesion assays revealed that high levels of M. pneumoniae-specific antibodies in nasal secretions of paediatric patients prevented the adhesion of M. pneumoniae to respiratory epithelial cells. CONCLUSIONS: Our study demonstrates that M. pneumoniae-specific mucosal antibodies protect against bacterial adhesion to respiratory epithelial cells, and are induced only during M. pneumoniae infection and not during asymptomatic carriage. This is strikingly different from carriage with bacteria such as Streptococcus pneumoniae where mucosal antibodies are induced by bacterial carriage.
Asunto(s)
Infecciones Comunitarias Adquiridas , Neumonía por Mycoplasma , Neumonía , Anticuerpos Antibacterianos , Estudios de Casos y Controles , Niño , Infecciones Comunitarias Adquiridas/microbiología , Humanos , Mycoplasma pneumoniaeRESUMEN
Antibody responses to Mycoplasma pneumoniae correlate with pulmonary M. pneumoniae clearance. However, M. pneumoniae-specific IgG antibodies can cross-react with the myelin glycolipid galactocerebroside (GalC) and cause neurological disorders. We assessed whether antiglycolipid antibody formation is part of the physiological immune response to M. pneumoniae We show that antibodies against M. pneumoniae proteins and glycolipids arise in serum of M. pneumoniae-infected children and mice. Although antibodies to M. pneumoniae glycolipids were mainly IgG, anti-GalC antibodies were only IgM. B-1a cells, shown to aid in protection against pathogen-derived glycolipids, are lacking in Bruton tyrosine kinase (Btk)-deficient mice. M. pneumoniae-infected Btk-deficient mice developed M. pneumoniae-specific IgG responses to M. pneumoniae proteins but not to M. pneumoniae glycolipids, including GalC. The equal recovery from M. pneumoniae infection in Btk-deficient and wild-type mice suggests that pulmonary M. pneumoniae clearance is predominantly mediated by IgG reactive with M. pneumoniae proteins and that M. pneumoniae glycolipid-specific IgG or IgM is not essential. These data will guide the development of M. pneumoniae-targeting vaccines that avoid the induction of neurotoxic antibodies.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/inmunología , Glucolípidos/inmunología , Mycoplasma pneumoniae/inmunología , Neumonía por Mycoplasma/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Niño , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , RatonesRESUMEN
Background: Carriage of Mycoplasma pneumoniae (Mp) in the nasopharynx is considered a prerequisite for pulmonary infection. It is interesting to note that Mp carriage is also detected after infection. Although B cells are known to be involved in pulmonary Mp clearance, their role in Mp carriage is unknown. Methods: In this study, we show in a mouse model that Mp persists in the nose after pulmonary infection, similar to humans. Results: Infection of mice enhanced Mp-specific immunoglobulin (Ig) M and IgG levels in serum and bronchoalveolar lavage fluid. However, nasal washes only contained elevated Mp-specific IgA. These differences in Ig compartmentalization correlated with differences in Mp-specific B cell responses between nose- and lung-draining lymphoid tissues. Moreover, transferred Mp-specific serum Igs had no effect on nasal carriage in B cell-deficient µMT mice, whereas this enabled µMT mice to clear pulmonary Mp infection. Conclusions: We report the first evidence that humoral immunity is limited in clearing Mp from the upper respiratory tract.
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Linfocitos B/inmunología , Portador Sano/inmunología , Mycoplasma pneumoniae/inmunología , Nasofaringe/inmunología , Nasofaringe/microbiología , Neumonía por Mycoplasma/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Inmunoglobulina A/análisis , Inmunoglobulina G/sangre , Ratones Endogámicos C57BL , Mucosa Nasal/inmunologíaRESUMEN
Detection of Mycoplasma pneumoniae by real-time PCR is not yet standardized across laboratories. We have implemented a standardization protocol to compare the performance of thirteen commercial and in-house approaches. Despite differences on threshold values of samples, all assays were able to detect at least 20M. pneumoniae genomes per reaction.
Asunto(s)
ADN Bacteriano/análisis , Genoma Bacteriano/genética , Mycoplasma pneumoniae/genética , Neumonía por Mycoplasma/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , ADN Bacteriano/genética , Humanos , Mycoplasma pneumoniae/aislamiento & purificación , Neumonía por Mycoplasma/microbiologíaRESUMEN
Mycoplasma pneumoniae is a common cause of respiratory tract infections (RTIs) in children. We recently demonstrated that this bacterium can be carried asymptomatically in the respiratory tract of children. To identify potential genetic differences between M. pneumoniae strains that are carried asymptomatically and those that cause symptomatic infections, we performed whole-genome sequence analysis of 20 M. pneumoniae strains. The analyzed strains included 3 reference strains, 3 strains isolated from asymptomatic children, 13 strains isolated from clinically well-defined patients suffering from an upper (n = 4) or lower (n = 9) RTI, and one strain isolated from a follow-up patient who recently recovered from an RTI. The obtained sequences were each compared to the sequences of the reference strains. To find differences between strains isolated from asymptomatic and symptomatic individuals, a variant comparison was performed between the different groups of strains. Irrespective of the group (asymptomatic vs. symptomatic) from which the strains originated, subtype 1 and subtype 2 strains formed separate clusters. We could not identify a specific genotype associated with M. pneumoniae virulence. However, we found marked genetic differences between clinical isolates and the reference strains, which indicated that the latter strains may not be regarded as appropriate representatives of circulating M. pneumoniae strains.
RESUMEN
OBJECTIVE: Guillain-Barré syndrome (GBS) is an acute postinfectious immune-mediated polyneuropathy. Although preceding respiratory tract infections with Mycoplasma pneumoniae have been reported in some cases, the role of M. pneumoniae in the pathogenesis of GBS remains unclear. We here cultured, for the first time, M. pneumoniae from a GBS patient with antibodies against galactocerebroside (GalC), which cross-reacted with the isolate. This case prompted us to unravel the role of M. pneumoniae in GBS in a case-control study. METHODS: We included 189 adults and 24 children with GBS and compared them to control cohorts for analysis of serum antibodies against M. pneumoniae (n = 479) and GalC (n = 198). RESULTS: Anti-M. pneumoniae immunoglobulin (Ig) M antibodies were detected in GBS patients and healthy controls in 3% and 0% of adults (p = 0.16) and 21% and 7% of children (p = 0.03), respectively. Anti-GalC antibodies (IgM and/or IgG) were found in 4% of adults and 25% of children with GBS (p = 0.001). Anti-GalC-positive patients showed more-frequent preceding respiratory symptoms, cranial nerve involvement, and a better outcome. Anti-GalC antibodies correlated with anti-M. pneumoniae antibodies (p < 0.001) and cross-reacted with different M. pneumoniae strains. Anti-GalC IgM antibodies were not only found in GBS patients with M. pneumoniae infection, but also in patients without neurological disease (8% vs 9%; p = 0.87), whereas anti-GalC IgG was exclusively found in patients with GBS (9% vs 0%; p = 0.006). INTERPRETATION: M. pneumoniae infection is associated with GBS, more frequently in children than adults, and elicits anti-GalC antibodies, of which specifically anti-GalC IgG may contribute to the pathogenesis of GBS. Ann Neurol 2016;80:566-580.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Autoanticuerpos/inmunología , Galactosilceramidas/inmunología , Síndrome de Guillain-Barré/inmunología , Infecciones por Mycoplasma/inmunología , Mycoplasma pneumoniae/inmunología , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Niño , Reacciones Cruzadas , Femenino , Síndrome de Guillain-Barré/etiología , Humanos , Inmunoglobulina G , Inmunoglobulina M , Masculino , Persona de Mediana Edad , Infecciones por Mycoplasma/complicaciones , Adulto JovenRESUMEN
The DNA recombination and repair machinery of Mycoplasma pneumoniae is composed of a limited set of approximately 11 proteins. Two of these proteins were predicted to be encoded by neighboring open reading frames (ORFs) MPN340 and MPN341. Both ORFs were found to have sequence similarity with genes that encode proteins belonging to the DNA helicase superfamily 1 (SF1). Interestingly, while a homolog of the MPN341 ORF is present in the genome of Mycoplasma genitalium (ORF MG244), MPN340 is an M. pneumoniae-specific ORF that is not found in other mycoplasmas. Moreover, the length of MPN340 (1590 base pairs [bp]) is considerably shorter than that of MPN341 (2148 bp). Examination of the MPN340-encoded amino acid sequence indicated that it may lack a so-called 2B subdomain, which is found in most SF1 DNA helicases. Also, the MPN340-encoded amino acid sequence was found to differ between subtype 1 strain M129 and subtype 2 strain FH at three amino acid positions. Both protein variants, which were termed PcrA(s) M129 and PcrA(s) FH, respectively, as well as the MPN341- and MG244-encoded proteins (PcrA Mpn and PcrA Mge , respectively), were purified, and tested for their ability to interact with DNA. While PcrA Mpn and PcrA Mge were found to bind preferentially to single-stranded DNA, both PcrA(s) M129 and PcrA(s) FH did not demonstrate significant DNA binding. However, all four proteins were found to have divalent cation- and ATP-dependent DNA helicase activity. The proteins displayed highest activity on partially double-stranded DNA substrates carrying 3' single-stranded extensions.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Mycoplasma genitalium/enzimología , Mycoplasma genitalium/genética , Mycoplasma pneumoniae/enzimología , Mycoplasma pneumoniae/genética , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , ADN Helicasas/química , ADN Helicasas/aislamiento & purificación , ADN Bacteriano/metabolismo , Orden Génico , Hidrólisis , Sistemas de Lectura Abierta , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: Mycoplasma pneumoniae is thought to be a common cause of respiratory tract infections (RTIs) in children. The diagnosis of M. pneumoniae RTIs currently relies on serological methods and/or the detection of bacterial DNA in the upper respiratory tract (URT). It is conceivable, however, that these diagnostic methods also yield positive results if M. pneumoniae is carried asymptomatically in the URT. Positive results from these tests may therefore not always be indicative of a symptomatic infection. The existence of asymptomatic carriage of M. pneumoniae has not been established. We hypothesized that asymptomatic carriage in children exists and investigated whether colonization and symptomatic infection could be differentiated by current diagnostic methods. METHODS AND FINDINGS: This study was conducted at the Erasmus MC-Sophia Children's Hospital and the after-hours General Practitioners Cooperative in Rotterdam, The Netherlands. Asymptomatic children (nâ=â405) and children with RTI symptoms (nâ=â321) aged 3 mo to 16 y were enrolled in a cross-sectional study from July 1, 2008, to November 30, 2011. Clinical data, pharyngeal and nasopharyngeal specimens, and serum samples were collected. The primary objective was to differentiate between colonization and symptomatic infection with M. pneumoniae by current diagnostic methods, especially real-time PCR. M. pneumoniae DNA was detected in 21.2% (95% CI 17.2%-25.2%) of the asymptomatic children and in 16.2% (95% CI 12.2%-20.2%) of the symptomatic children (pâ=â0.11). Neither serology nor quantitative PCR nor culture differentiated asymptomatic carriage from infection. A total of 202 children were tested for the presence of other bacterial and viral pathogens. Two or more pathogens were found in 56% (63/112) of the asymptomatic children and in 55.5% (50/90) of the symptomatic children. Finally, longitudinal sampling showed persistence of M. pneumoniae in the URT for up to 4 mo. Fifteen of the 21 asymptomatic children with M. pneumoniae and 19 of the 22 symptomatic children with M. pneumoniae in this longitudinal follow-up tested negative after 1 mo. CONCLUSIONS: Although our study has limitations, such as a single study site and limited sample size, our data indicate that the presence of M. pneumoniae in the URT is common in asymptomatic children. The current diagnostic tests for M. pneumoniae are unable to differentiate between asymptomatic carriage and symptomatic infection.
Asunto(s)
Portador Sano , Mycoplasma pneumoniae/patogenicidad , Neumonía por Mycoplasma/microbiología , Neumonía por Mycoplasma/transmisión , Sistema Respiratorio/microbiología , Adolescente , Anticuerpos Antibacterianos/sangre , Enfermedades Asintomáticas , Técnicas Bacteriológicas , Distribución de Chi-Cuadrado , Niño , Preescolar , Estudios Transversales , ADN Bacteriano/aislamiento & purificación , Diagnóstico Diferencial , Femenino , Humanos , Lactante , Modelos Logísticos , Masculino , Análisis Multivariante , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/inmunología , Mycoplasma pneumoniae/aislamiento & purificación , Países Bajos , Oportunidad Relativa , Neumonía por Mycoplasma/sangre , Neumonía por Mycoplasma/diagnóstico , Valor Predictivo de las Pruebas , Reacción en Cadena en Tiempo Real de la Polimerasa , Pruebas Serológicas , Factores de TiempoRESUMEN
The DNA recombination and repair machineries of Mycoplasma genitalium and Mycoplasma pneumoniae differ considerably from those of gram-positive and gram-negative bacteria. Most notably, M. pneumoniae is unable to express a functional RecU Holliday junction (HJ) resolvase. In addition, the RuvB homologues from both M. pneumoniae and M. genitalium only exhibit DNA helicase activity but not HJ branch migration activity in vitro. To identify a putative role of the RuvA homologues of these mycoplasmas in DNA recombination, both proteins (RuvA(Mpn) and RuvA(Mge), respectively) were studied for their ability to bind DNA and to interact with RuvB and RecU. In spite of a high level of sequence conservation between RuvA(Mpn) and RuvA(Mge) (68.8% identity), substantial differences were found between these proteins in their activities. First, RuvA(Mge) was found to preferentially bind to HJs, whereas RuvA(Mpn) displayed similar affinities for both HJs and single-stranded DNA. Second, while RuvA(Mpn) is able to form two distinct complexes with HJs, RuvA(Mge) only produced a single HJ complex. Third, RuvA(Mge) stimulated the DNA helicase and ATPase activities of RuvB(Mge), whereas RuvA(Mpn) did not augment RuvB activity. Finally, while both RuvA(Mge) and RecU(Mge) efficiently bind to HJs, they did not compete with each other for HJ binding, but formed stable complexes with HJs over a wide protein concentration range. This interaction, however, resulted in inhibition of the HJ resolution activity of RecU(Mge).
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Mycoplasma genitalium/metabolismo , Neumonía por Mycoplasma/metabolismo , Homología de Secuencia de Aminoácido , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Cruciforme/genética , ADN Cruciforme/metabolismo , Datos de Secuencia Molecular , Mycoplasma genitalium/genética , Oligodesoxirribonucleótidos/metabolismo , Sistemas de Lectura Abierta/genética , Neumonía por Mycoplasma/genética , Unión Proteica , Recombinación GenéticaRESUMEN
An important role in the treatment regimens for Mycoplasma pneumoniae infections is played by macrolide (ML) antibiotics. In the past few years, however, a steady increase has been detected in the worldwide prevalence of ML-resistant (ML(r)) M. pneumoniae strains. It is obvious that this increase necessitates a continuous monitoring of ML(r) and, when detected, modification of antibiotic treatment modalities. Previously, we developed a pyrosequencing-based assay system for the genetic determination of ML(r) as well as molecular typing of M. pneumoniae. In this study, the sensitivity of this system was improved by the inclusion of a nested-PCR protocol. The modified system was applied to 114 M. pneumoniae-positive specimens that were obtained from a collection of 4,390 samples from patients with acute respiratory tract infections. These samples were collected between 1997 and 2008 in The Netherlands. The pyrosequencing system produced reliable data in 86% of the specimens that contained >500 M. pneumoniae genome copies/ml of patient sample. Each of these samples contained DNA of the ML-sensitive genotype. While 43% of the samples were found to harbor the M. pneumoniae subtype 1 genotype, 57% contained the subtype 2 genotype. We conclude that the pyrosequencing-based assay system is a useful tool for ML(r) determination and molecular typing of M. pneumoniae in patient samples. ML(r)-associated M. pneumoniae genotypes, however, were not found in the current study population.
Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana , Macrólidos/farmacología , Tipificación Molecular/métodos , Mycoplasma pneumoniae/clasificación , Mycoplasma pneumoniae/efectos de los fármacos , Neumonía por Mycoplasma/microbiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Pruebas de Sensibilidad Microbiana/métodos , Persona de Mediana Edad , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/aislamiento & purificación , Países Bajos , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Adulto JovenRESUMEN
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.
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Adhesinas Bacterianas/genética , Mycoplasma pneumoniae/genética , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Clonación Molecular , ADN Bacteriano/genética , Variación Genética , Genotipo , Análisis de Secuencia de ADNRESUMEN
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 (RecU(Mge) and RecU(Mpn) respectively), which share sequence similarity with RecU Holliday junction (HJ) resolvases. RecU(Mge) was found to: (i) bind HJ substrates and large double-stranded DNA molecules and (ii) cleave HJ substrates at the sequence 5'-(G) /(T) C↓(C) /(T) T(A) /(G) G-3' in the presence of Mn(2+). Interestingly, RecU(Mpn) (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 RecU(Mge). Additionally, RecU(Mpn) appears 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 RecU(Mge) is a HJ resolvase that may play a central role in recombination in M. genitalium.
Asunto(s)
Resolvasas de Unión Holliday/genética , Resolvasas de Unión Holliday/metabolismo , Mycoplasma genitalium/enzimología , Mycoplasma genitalium/genética , Mycoplasma pneumoniae/enzimología , Mycoplasma pneumoniae/genética , Secuencia de Bases , Coenzimas/metabolismo , ADN Bacteriano/metabolismo , Manganeso/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Farmacorresistencia Bacteriana , Macrólidos/farmacología , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/aislamiento & purificación , Neumonía por Mycoplasma/microbiología , Análisis de Secuencia de ADN/métodos , Antibacterianos/farmacología , Secuencia de Bases , ADN Bacteriano/genética , ADN Ribosómico/genética , Humanos , Datos de Secuencia Molecular , Mycoplasma pneumoniae/clasificación , Mycoplasma pneumoniae/efectos de los fármacos , Polimorfismo de Longitud del Fragmento de Restricción , ARN Ribosómico 23S/genéticaRESUMEN
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.
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Adhesinas Bacterianas/genética , Variación Genética , Mycoplasma pneumoniae/genética , Recombinación Genética , Secuencia de Bases , ADN Bacteriano/análisis , ADN Bacteriano/genética , Evolución Molecular , Genoma Bacteriano , Datos de Secuencia Molecular , Mycoplasma pneumoniae/clasificación , Análisis de Secuencia de ADNRESUMEN
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 approximately 76 % isolates were of subtype 1, approximately 20 % of subtype 2 and approximately 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.
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Técnicas de Tipificación Bacteriana/métodos , Carotenoides/química , Mycoplasma pneumoniae/química , Neumonía por Mycoplasma/microbiología , Espectrometría Raman , Adhesinas Bacterianas/genética , Secuencia de Aminoácidos , Carotenoides/biosíntesis , ADN Bacteriano/análisis , ADN Bacteriano/genética , Variación Genética , Humanos , Redes y Vías Metabólicas , Datos de Secuencia Molecular , Mycoplasma pneumoniae/clasificación , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Portador Sano/inmunología , Nariz/microbiología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Anticuerpos Antibacterianos/sangre , Especificidad de Anticuerpos , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Enterotoxinas/inmunología , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Pruebas de Neutralización , Reproducibilidad de los Resultados , Superantígenos/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Mycoplasma pneumoniae/genética , Rec A Recombinasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , ADN Bacteriano/genética , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Mycoplasma pneumoniae/metabolismo , Sistemas de Lectura Abierta , Unión Proteica , Rec A Recombinasas/genética , Rec A Recombinasas/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Recombinación Genética , Alineación de Secuencia , Especificidad por SustratoRESUMEN
CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.
Asunto(s)
Proteínas Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica , Streptococcus pneumoniae/fisiología , Factores de Transcripción/fisiología , Aminoácidos/metabolismo , Animales , Adhesión Bacteriana/genética , Sitios de Unión , Carbono/metabolismo , Huella de ADN , ADN Bacteriano/metabolismo , Electroforesis en Gel Bidimensional , Ensayo de Cambio de Movilidad Electroforética , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Hierro/metabolismo , Redes y Vías Metabólicas/genética , Ratones , Mutagénesis Insercional , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones Neumocócicas/microbiología , Unión Proteica , Proteoma/análisis , Streptococcus pneumoniae/patogenicidad , Factores de Transcripción/genética , Virulencia/genéticaRESUMEN
Streptococcus pneumoniae expresses two surface-exposed lipoproteins, PpmA and SlrA, which share homology with distinct families of peptidyl-prolyl isomerases (PPIases). In this study, we demonstrated for the first time that the lipoprotein cyclophilin, SlrA, can catalyze the cis-trans isomerization of proline containing tetrapeptides and that SlrA contributes to pneumococcal colonization. The substrate specificity of SlrA is typical for prokaryotic and eukaryotic cyclophilins, with Suc-Ala-Ala-Pro-Phe-p-nitroanilide (pNA) being the most rapidly catalyzed substrate. In a mouse pneumonia model the slrA knock-out D39DeltaslrA did not cause significant differences in the survival times of mice compared with the isogenic wild-type strain. In contrast, a detailed analysis of bacterial outgrowth over time in the nasopharynx, airways, lungs, blood, and spleen showed a rapid elimination of slrA mutants from the upper airways but did not reveal significant differences in the lungs, blood, and spleen. These results suggested that SlrA is involved in colonization but does not contribute significantly to invasive pneumococcal disease. In cell culture infection experiments, the absence of SlrA impaired adherence to pneumococcal disease-specific epithelial and endothelial non-professional cell lines. Adherence of the slrA mutant could not be restored by exogenously added SlrA. Strikingly, deficiency in SlrA did not reduce binding activity to host target proteins, but resulted in enhanced uptake by professional phagocytes. In conclusion, SlrA is a functional, cyclophilin-type PPIase and contributes to pneumococcal virulence in the first stage of infection, namely, colonization of the upper airways, most likely by modulating the biological function of important virulence proteins.
Asunto(s)
Isomerasa de Peptidilprolil/química , Streptococcus pneumoniae/patogenicidad , Secuencia de Aminoácidos , Animales , Adhesión Celular , Línea Celular Tumoral , Ciclofilina A/química , Ciclofilinas/química , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Matriz Extracelular/metabolismo , Humanos , Lipoproteínas/química , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Péptidos/química , Isomerasa de Peptidilprolil/fisiología , Fagocitosis , Neumonía/microbiología , Prolina/química , Unión Proteica , Homología de Secuencia de Aminoácido , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Especificidad por Sustrato , Factores de Tiempo , Factores de Virulencia/metabolismoRESUMEN
Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.