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1.  Proteomics and Pathway Analyses of the Milk Fat Globule in Sheep Naturally Infected by Mycoplasma agalactiae Provide Indications of the In Vivo Response of the Mammary Epithelium to Bacterial Infection ▿ ‡  
Infection and Immunity  2011;79(9):3833-3845.
Milk fat globules (MFGs) are vesicles released in milk as fat droplets surrounded by the endoplasmic reticulum and apical cell membranes. During formation and apocrine secretion by lactocytes, various amounts of cytoplasmic crescents remain trapped within the released vesicle, making MFGs a natural sampling mechanism of the lactating cell contents. With the aim of investigating the events occurring in the mammary epithelium during bacterial infection, the MFG proteome was characterized by two-dimensional difference gel electrophoresis (2-D DIGE), SDS-PAGE followed by shotgun liquid chromatography-tandem mass spectrometry (GeLC-MS/MS), label-free quantification by the normalized spectral abundance factor (NSAF) approach, Western blotting, and pathway analysis, using sheep naturally infected by Mycoplasma agalactiae. A number of protein classes were found to increase in MFGs upon infection, including proteins involved in inflammation and host defense, cortical cytoskeleton proteins, heat shock proteins, and proteins related to oxidative stress. Conversely, a strikingly lower abundance was observed for proteins devoted to MFG metabolism and secretion. To our knowledge, this is the first report describing proteomic changes occurring in MFGs during sheep infectious mastitis. The results presented here offer new insights into the in vivo response of mammary epithelial cells to bacterial infection and open the way to the discovery of protein biomarkers for monitoring clinical and subclinical mastitis.
doi:10.1128/IAI.00040-11
PMCID: PMC3165467  PMID: 21690237
2.  Critical Role of Dispensable Genes in Mycoplasma agalactiae Interaction with Mammalian Cells▿  
Infection and Immunity  2010;78(4):1542-1551.
Mycoplasmas are minimal bacteria whose genomes barely exceed the smallest amount of information required to sustain autonomous life. Despite this apparent simplicity, several mycoplasmas are successful pathogens of humans and animals, in which they establish intimate interactions with epithelial cells at mucosal surfaces. To identify biological functions mediating mycoplasma interactions with mammalian cells, we produced a library of transposon knockout mutants in the ruminant pathogen Mycoplasma agalactiae and used this library to identify mutants displaying a growth-deficient pheonotype in cell culture. M. agalactiae mutants displaying a 3-fold reduction in CFU titers to nearly complete extinction in coculture with HeLa cells were identified. Mapping of transposon insertion sites revealed 18 genomic regions putatively involved in the interaction of M. agalactiae with HeLa cells. Several of these regions encode proteins with features of membrane lipoproteins and/or were involved in horizontal gene transfer with phylogenetically distant pathogenic mycoplasmas of ruminants. Two mutants with the most extreme phenotype carry a transposon in a genomic region designated the NIF locus which encodes homologues of SufS and SufU, two proteins presumably involved in [Fe-S] cluster biosynthesis in Gram-positive bacteria. Complementation studies confirmed the conditional essentiality of the NIF locus, which was found to be critical for proliferation in the presence of HeLa cells and several other mammalian cell lines but dispensable for axenic growth. While our results raised questions regarding essential functions in mycoplasmas, they also provide a means for studying the role of mycoplasmas as minimal pathogens.
doi:10.1128/IAI.01195-09
PMCID: PMC2849427  PMID: 20123713
3.  Being Pathogenic, Plastic, and Sexual while Living with a Nearly Minimal Bacterial Genome 
PLoS Genetics  2007;3(5):e75.
Mycoplasmas are commonly described as the simplest self-replicating organisms, whose evolution was mainly characterized by genome downsizing with a proposed evolutionary scenario similar to that of obligate intracellular bacteria such as insect endosymbionts. Thus far, analysis of mycoplasma genomes indicates a low level of horizontal gene transfer (HGT) implying that DNA acquisition is strongly limited in these minimal bacteria. In this study, the genome of the ruminant pathogen Mycoplasma agalactiae was sequenced. Comparative genomic data and phylogenetic tree reconstruction revealed that ∼18% of its small genome (877,438 bp) has undergone HGT with the phylogenetically distinct mycoides cluster, which is composed of significant ruminant pathogens. HGT involves genes often found as clusters, several of which encode lipoproteins that usually play an important role in mycoplasma–host interaction. A decayed form of a conjugative element also described in a member of the mycoides cluster was found in the M. agalactiae genome, suggesting that HGT may have occurred by mobilizing a related genetic element. The possibility of HGT events among other mycoplasmas was evaluated with the available sequenced genomes. Our data indicate marginal levels of HGT among Mycoplasma species except for those described above and, to a lesser extent, for those observed in between the two bird pathogens, M. gallisepticum and M. synoviae. This first description of large-scale HGT among mycoplasmas sharing the same ecological niche challenges the generally accepted evolutionary scenario in which gene loss is the main driving force of mycoplasma evolution. The latter clearly differs from that of other bacteria with small genomes, particularly obligate intracellular bacteria that are isolated within host cells. Consequently, mycoplasmas are not only able to subvert complex hosts but presumably have retained sexual competence, a trait that may prevent them from genome stasis and contribute to adaptation to new hosts.
Author Summary
Mycoplasmas are cell wall–lacking prokaryotes that evolved from ancestors common to Gram-positive bacteria by way of massive losses of genetic material. With their minimal genome, mycoplasmas are considered to be the simplest free-living organisms, yet several species are successful pathogens of man and animal. In this study, we challenged the commonly accepted view in which mycoplasma evolution is driven only by genome down-sizing. Indeed, we showed that a significant amount of genes underwent horizontal transfer among different mycoplasma species that share the same ruminant hosts. In these species, the occurrence of a genetic element that can promote DNA transfer via cell-to-cell contact suggests that some mycoplasmas may have retained or acquired sexual competence. Transferred genes were found to encode proteins that are likely to be associated with mycoplasma–host interactions. Sharing genetic resources via horizontal gene transfer may provide mycoplasmas with a means for adapting to new niches or to new hosts and for avoiding irreversible genome erosion.
doi:10.1371/journal.pgen.0030075
PMCID: PMC1868952  PMID: 17511520
4.  Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro- and micro-events that are shaping mycoplasma diversity 
BMC Genomics  2010;11:86.
Background
While the genomic era is accumulating a tremendous amount of data, the question of how genomics can describe a bacterial species remains to be fully addressed. The recent sequencing of the genome of the Mycoplasma agalactiae type strain has challenged our general view on mycoplasmas by suggesting that these simple bacteria are able to exchange significant amount of genetic material via horizontal gene transfer. Yet, events that are shaping mycoplasma genomes and that are underlining diversity within this species have to be fully evaluated. For this purpose, we compared two strains that are representative of the genetic spectrum encountered in this species: the type strain PG2 which genome is already available and a field strain, 5632, which was fully sequenced and annotated in this study.
Results
The two genomes differ by ca. 130 kbp with that of 5632 being the largest (1006 kbp). The make up of this additional genetic material mainly corresponds (i) to mobile genetic elements and (ii) to expanded repertoire of gene families that encode putative surface proteins and display features of highly-variable systems. More specifically, three entire copies of a previously described integrative conjugative element are found in 5632 that accounts for ca. 80 kbp. Other mobile genetic elements, found in 5632 but not in PG2, are the more classical insertion sequences which are related to those found in two other ruminant pathogens, M. bovis and M. mycoides subsp. mycoides SC. In 5632, repertoires of gene families encoding surface proteins are larger due to gene duplication. Comparative proteomic analyses of the two strains indicate that the additional coding capacity of 5632 affects the overall architecture of the surface and suggests the occurrence of new phase variable systems based on single nucleotide polymorphisms.
Conclusion
Overall, comparative analyses of two M. agalactiae strains revealed a very dynamic genome which structure has been shaped by gene flow among ruminant mycoplasmas and expansion-reduction of gene repertoires encoding surface proteins, the expression of which is driven by localized genetic micro-events.
doi:10.1186/1471-2164-11-86
PMCID: PMC2824730  PMID: 20122262
5.  Characterization of P40, a Cytadhesin of Mycoplasma agalactiae  
Infection and Immunity  2002;70(10):5612-5621.
An immunodominant protein, P40, of Mycoplasma agalactiae was analyzed genetically and functionally. The gene encoding P40 was cloned from type strain PG2, sequenced, submitted to point mutagenesis in order to convert mycoplasma-specific TGATrp codon to the universal TGGTrp codon, and subsequently expressed in Escherichia coli. Nucleotide sequence-derived amino acid sequence comparisons revealed a similarity of P40 to the adhesin P50 of Mycoplasma hominis and to protein P89 of Spiroplasma citri, which is expected to be involved in adhesion. The amino acid sequence of P40 revealed a recognition site for a signal peptidase and strong antigenic and hydrophilic motifs in the C-terminal domain. Triton X-114 phase partitioning confirmed that P40 is a membrane protein. Fab fragments of antibodies directed against recombinant purified P40 significantly inhibited adherence of M. agalactiae strains PG2 to lamb joint synovial cells LSM 192. Sera taken sequentially from sheep infected with PG2 revealed that P40 induced a strong and persistent immune response that gave strong signals on immunoblots containing recombinant P40 even 3 months after infection. The gene encoding P40 was present in a single copy in all of the 26 field strains of M. agalactiae analyzed and was not detected in closely related mycoplasma species. P40 was expressed as a protein with an apparent molecular mass of 37 kDa on sodium dodecyl sulfate-acrylamide gels by all M. agalactiae strains except for serotype C strains, which showed nonsense mutations in their p40 genes.
doi:10.1128/IAI.70.10.5612-5621.2002
PMCID: PMC128363  PMID: 12228289
6.  Mycoplasma agalactiae, an Etiological Agent of Contagious Agalactia in Small Ruminants: A Review 
Mycoplasma agalactiae is one of the causal agents of classical contagious agalactia (CA), a serious, economically important but neglected enzootic disease of small ruminants. It occurs in many parts of the world and most notably in the Mediterranean Basin. Following the infection common complications are septicaemia, mastitis, arthritis, pleurisy, pneumonia, and keratoconjunctivitis. Primary or tentative diagnosis of the organism is based upon clinical signs. Various serological tests, namely, growth precipitation, immunofluorescence, complement fixation test, haemagglutination inhibition, agglutination, immunodiffusion, enzyme immunoassays, immunoelectrophoresis, blotting techniques, and others, are available. Molecular tools seem to be much more sensitive, specific, and faster and help to differentiate various strains. The real-time PCR, multiplex PCR, quantitative PCR, PCR-RFLP, MLST, and gene probes, complementary to segments of chromosomal DNA or 16S ribosomal RNA (rRNA), have strengthened the diagnosis of M. agalactiae. Both live attenuated and adjuvant (alum precipitated or saponified) inactivated vaccines are available with greater use of inactivated ones due to lack of side effects. The present review discusses the etiology, epidemiology, pathogenesis, and clinical signs of contagious agalactia in small ruminants along with trends and advances in its diagnosis, treatment, vaccination, prevention, and control strategies that will help in countering this disease.
doi:10.1155/2014/286752
PMCID: PMC4109668  PMID: 25097796
7.  Characterization and Analysis of a Stable Serotype-Associated Membrane Protein (P30) of Mycoplasma agalactiae 
Journal of Clinical Microbiology  2001;39(8):2814-2822.
The gene for a 30-kDa immunodominant antigen, P30, of Mycoplasma agalactiae was cloned from type strain PG2 and expressed in Escherichia coli. P30 is encoded on a monocistronic operon determined by two −10 boxes and a possible −35 region constituting the potential promoter, and a transcription termination site. The gene for the 266-amino-acid protein is preceded by a polypurine-rich region designed as the consensus sequence for a ribosome-binding site. Analysis of the amino acid sequence of P30 revealed the presence of a recognition site for a prokaryotic signal peptidase II at amino acid (aa) 24, indicating that P30 is a transmembrane protein. Moreover, Triton X-114 phase partitioning of M. agalactiae PG2 total antigen revealed that P30 is strongly hydrophobic and hence a possible membrane component. Immunoblot analysis using the monospecific polyclonal anti-P30-His serum indicated that P30 is specific to M. agalactiae. Furthermore, PCR amplification with specific primers for p30 and Southern blot analysis revealed the presence of the gene in all M. agalactiae strains tested and its absence in the other mycoplasma species. Among 27 strains of M. agalactiae studied, 20 strains belonging to the common serotypes A to D, including PG2, expressed P30 or part of it as detected by the monospecific polyclonal anti-P30 antibodies. The other seven strains belonging to the rarely isolated serotypes E to H were negative for P30. The p30 gene was sequenced in 15 strains of M. agalactiae, 10 of which expressed P30 or at least part of it and 5 of which did not express P30. The negative strains carried mutations in both −10 boxes of the promoters. These mutations seem to be responsible for the lack of P30 expression in these strains. Analysis of sera from sheep that were experimentally infected with M. agalactiae revealed that P30 induced a strong and persistent immune response which was still very high two months after infection. In contrast, currently used enzyme-linked immunosorbent assay serology gave only low titers.
doi:10.1128/JCM.39.8.2814-2822.2001
PMCID: PMC88244  PMID: 11473997
8.  The Novel Fibrinogen-Binding Protein FbsB Promotes Streptococcus agalactiae Invasion into Epithelial Cells  
Infection and Immunity  2004;72(6):3495-3504.
Streptococcus agalactiae is a major cause of bacterial sepsis and meningitis in human newborns. The interaction of S. agalactiae with host proteins and the entry into host cells thereby represent important virulence traits of these bacteria. The present report describes the identification of the fbsB gene, encoding a novel fibrinogen-binding protein that plays a crucial role in the invasion of S. agalactiae into human cells. In Western blots and enzyme-linked immunosorbent assay (ELISA) experiments, the FbsB protein was demonstrated to interact with soluble and immobilized fibrinogen. Binding studies showed the N-terminal 388 residues of FbsB and the Aα-subunit of human fibrinogen to recognize each other. By reverse transcription (RT)-PCR, the fbsB gene was shown to be cotranscribed with the gbs0851 gene in S. agalactiae. Deletion of the fbsB gene in the genome of S. agalactiae did not influence the binding of the bacteria to fibrinogen, suggesting that FbsB does not participate in the attachment of S. agalactiae to fibrinogen. In tissue culture experiments, however, the fbsB deletion mutant was severely impaired in its invasion into lung epithelial cells. Bacterial invasion could be reestablished by introducing the fbsB gene on a shuttle plasmid into the fbsB deletion mutant. Furthermore, treatment of lung epithelial cells with FbsB fusion protein blocked S. agalactiae invasion of epithelial cells in a dose-dependent fashion. These results suggest an important role of the FbsB protein in the overall process of host cell entry by S. agalactiae.
doi:10.1128/IAI.72.6.3495-3504.2004
PMCID: PMC415667  PMID: 15155657
9.  Genome-Scale Analysis of Mycoplasma agalactiae Loci Involved in Interaction with Host Cells 
PLoS ONE  2011;6(9):e25291.
Mycoplasma agalactiae is an important pathogen of small ruminants, in which it causes contagious agalactia. It belongs to a large group of “minimal bacteria” with a small genome and reduced metabolic capacities that are dependent on their host for nutrients. Mycoplasma survival thus relies on intimate contact with host cells, but little is known about the factors involved in these interactions or in the more general infectious process. To address this issue, an assay based on goat epithelial and fibroblastic cells was used to screen a M. agalactiae knockout mutant library. Mutants with reduced growth capacities in cell culture were selected and 62 genomic loci were identified as contributing to this phenotype. As expected for minimal bacteria, “transport and metabolism” was the functional category most commonly implicated in this phenotype, but 50% of the selected mutants were disrupted in coding sequences (CDSs) with unknown functions, with surface lipoproteins being most commonly represented in this category. Since mycoplasmas lack a cell wall, lipoproteins are likely to be important in interactions with the host. A few intergenic regions were also identified that may act as regulatory sequences under co-culture conditions. Interestingly, some mutants mapped to gene clusters that are highly conserved across mycoplasma species but located in different positions. One of these clusters was found in a transcriptionally active region of the M. agalactiae chromosome, downstream of a cryptic promoter. A possible scenario for the evolution of these loci is discussed. Finally, several CDSs identified here are conserved in other important pathogenic mycoplasmas, and some were involved in horizontal gene transfer with phylogenetically distant species. These results provide a basis for further deciphering functions mediating mycoplasma-host interactions.
doi:10.1371/journal.pone.0025291
PMCID: PMC3179502  PMID: 21966487
10.  Evaluation of a Novel Chimeric B Cell Epitope-Based Vaccine against Mastitis Induced by Either Streptococcus agalactiae or Staphylococcus aureus in Mice▿ 
To construct a universal vaccine against mastitis induced by either Streptococcus agalactiae or Staphylococcus aureus, the B cell epitopes of the surface immunogenic protein (Sip) from S. agalactiae and clumping factor A (ClfA) from S. aureus were analyzed and predicted. sip-clfA, a novel chimeric B cell epitope-based gene, was obtained by overlap PCR, and then the recombinant Sip-ClfA (rSip-ClfA) was expressed and purified. rSip-ClfA and inactivated S. agalactiae and S. aureus were formulated into different vaccines with mineral oil as the adjuvant and evaluated in mouse models. The rSip-ClfA vaccination induced immunoglobulin G (IgG) titers higher than those seen in groups immunized with inactivated bacteria. Furthermore, the response to rSip-ClfA immunization was characterized as having a dominant IgG1 subtype, whereas both bacterial immunizations produced similar levels of IgG1 and IgG2a. The antiserum capacities for opsonizing adhesion and phagocytosis were significantly greater in the rSip-ClfA immunization group than in the killed-bacterium immunization groups (P < 0.05). The immunized lactating mice were challenged with either S. agalactiae or S. aureus via the intramammary route. At 24 h postinfection, the numbers of bacteria recovered from the mammary glands in the rSip-ClfA group were >5-fold lower than those in both inactivated-bacterium groups (P < 0.01). Histopathological examination of the mammary glands showed that rSip-ClfA immunization provided better protection of mammary gland tissue integrity against both S. agalactiae and S. aureus challenges. Thus, the recombinant protein rSip-ClfA would be a promising vaccine candidate against mastitis induced by either S. agalactiae or S. aureus.
doi:10.1128/CVI.00066-11
PMCID: PMC3122606  PMID: 21508165
11.  Mycoplasma agalactiae MAG_5040 is a Mg2+-Dependent, Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural Infection 
PLoS ONE  2013;8(2):e57775.
In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl2 at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.
doi:10.1371/journal.pone.0057775
PMCID: PMC3585158  PMID: 23469065
12.  Absolute quantification of microbial proteomes at different states by directed mass spectrometry 
The developed, directed mass spectrometry workflow allows to generate consistent and system-wide quantitative maps of microbial proteomes in a single analysis. Application to the human pathogen L. interrogans revealed mechanistic proteome changes over time involved in pathogenic progression and antibiotic defense, and new insights about the regulation of absolute protein abundances within operons.
The developed, directed proteomic approach allowed consistent detection and absolute quantification of 1680 proteins of the human pathogen L. interrogans in a single LC–MS/MS experiment.The comparison of 25 extensive, consistent and quantitative proteome maps revealed new insights about the proteome changes involved in pathogenic progression and antibiotic defense of L. interrogans, and about the regulation of protein abundances within operons.The generated time-resolved data sets are compatible with pattern analysis algorithms developed for transcriptomics, including hierarchical clustering and functional enrichment analysis of the detected profile clusters.This is the first study that describes the absolute quantitative behavior of any proteome over multiple states and represents the most comprehensive proteome abundance pattern comparison for any organism to date.
Over the last decade, mass spectrometry (MS)-based proteomics has evolved as the method of choice for system-wide proteome studies and now allows for the characterization of several thousands of proteins in a single sample. Despite these great advances, redundant monitoring of protein levels over large sample numbers in a high-throughput manner remains a challenging task. New directed MS strategies have shown to overcome some of the current limitations, thereby enabling the acquisition of consistent and system-wide data sets of proteomes with low-to-moderate complexity at high throughput.
In this study, we applied this integrated, two-stage MS strategy to investigate global proteome changes in the human pathogen L. interrogans. In the initial discovery phase, 1680 proteins (out of around 3600 gene products) could be identified (Schmidt et al, 2008) and, by focusing precious MS-sequencing time on the most dominant, specific peptides per protein, all proteins could be accurately and consistently monitored over 25 different samples within a few days of instrument time in the following scoring phase (Figure 1). Additionally, the co-analysis of heavy reference peptides enabled us to obtain absolute protein concentration estimates for all identified proteins in each perturbation (Malmström et al, 2009). The detected proteins did not show any biases against functional groups or protein classes, including membrane proteins, and span an abundance range of more than three orders of magnitude, a range that is expected to cover most of the L. interrogans proteome (Malmström et al, 2009).
To elucidate mechanistic proteome changes over time involved in pathogenic progression and antibiotic defense of L. interrogans, we generated time-resolved proteome maps of cells perturbed with serum and three different antibiotics at sublethal concentrations that are currently used to treat Leptospirosis. This yielded an information-rich proteomic data set that describes, for the first time, the absolute quantitative behavior of any proteome over multiple states, and represents the most comprehensive proteome abundance pattern comparison for any organism to date. Using this unique property of the data set, we could quantify protein components of entire pathways across several time points and subject the data sets to cluster analysis, a tool that was previously limited to the transcript level due to incomplete sampling on protein level (Figure 4). Based on these analyses, we could demonstrate that Leptospira cells adjust the cellular abundance of a certain subset of proteins and pathways as a general response to stress while other parts of the proteome respond highly specific. The cells furthermore react to individual treatments by ‘fine tuning' the abundance of certain proteins and pathways in order to cope with the specific cause of stress. Intriguingly, the most specific and significant expression changes were observed for proteins involved in motility, tissue penetration and virulence after serum treatment where we tried to simulate the host environment. While many of the detected protein changes demonstrate good agreement with available transcriptomics data, most proteins showed a poor correlation. This includes potential virulence factors, like Loa22 or OmpL1, with confirmed expression in vivo that were significantly up-regulated on the protein level, but not on the mRNA level, strengthening the importance of proteomic studies. The high resolution and coverage of the proteome data set enabled us to further investigate protein abundance changes of co-regulated genes within operons. This suggests that although most proteins within an operon respond to regulation synchronously, bacterial cells seem to have subtle means to adjust the levels of individual proteins or protein groups outside of the general trend, a phenomena that was recently also observed on the transcript level of other bacteria (Güell et al, 2009).
The method can be implemented with standard high-resolution mass spectrometers and software tools that are readily available in the majority of proteomics laboratories. It is scalable to any proteome of low-to-medium complexity and can be extended to post-translational modifications or peptide-labeling strategies for quantification. We therefore expect the approach outlined here to become a cornerstone for microbial systems biology.
Over the past decade, liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has evolved into the main proteome discovery technology. Up to several thousand proteins can now be reliably identified from a sample and the relative abundance of the identified proteins can be determined across samples. However, the remeasurement of substantially similar proteomes, for example those generated by perturbation experiments in systems biology, at high reproducibility and throughput remains challenging. Here, we apply a directed MS strategy to detect and quantify sets of pre-determined peptides in tryptic digests of cells of the human pathogen Leptospira interrogans at 25 different states. We show that in a single LC–MS/MS experiment around 5000 peptides, covering 1680 L. interrogans proteins, can be consistently detected and their absolute expression levels estimated, revealing new insights about the proteome changes involved in pathogenic progression and antibiotic defense of L. interrogans. This is the first study that describes the absolute quantitative behavior of any proteome over multiple states, and represents the most comprehensive proteome abundance pattern comparison for any organism to date.
doi:10.1038/msb.2011.37
PMCID: PMC3159967  PMID: 21772258
absolute quantification; directed mass spectrometry; Leptospira interrogans; microbiology; proteomics
13.  Analysis of RogB-Controlled Virulence Mechanisms and Gene Expression in Streptococcus agalactiae  
Infection and Immunity  2003;71(9):5056-5064.
Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis in neonates and also the causative agent of different serious infections in immunocompromised adults. The wide range of diseases that are caused by S. agalactiae suggests regulatory mechanisms that control the formation of specific virulence factors in these bacteria. The present study describes a gene from S. agalactiae, designated rogB, encoding a protein with significant similarity to members of the RofA-like protein (RALP) family of transcriptional regulators. Disruption of the rogB gene in the genome of S. agalactiae resulted in mutant strain RGB1, which was impaired in its ability to bind to fibrinogen and fibronectin. Mutant RGB1 also exhibited a reduced adherence to human epithelial cells but did not show an altered invasion of eukaryotic cells. By real-time PCR analysis, mutant RGB1 revealed an increased expression of the cpsA gene, encoding a regulator of capsule gene expression. However, strain RGB1 exhibited a reduced expression of the rogB gene and of two adjacent genes, encoding putative virulence factors in S. agalactiae. Furthermore, mutant RGB1 was impaired in the expression of the fbsA gene, coding for a fibrinogen receptor from S. agalactiae. The altered gene expression in mutant RGB1 could be restored by plasmid-mediated expression of rogB, confirming a RogB deficiency as the cause for the observed changes in virulence gene expression in S. agalactiae. Reporter gene studies with a promotorless luciferase gene fused to fbsA allowed a growth-dependent analysis of fbsA expression in S. agalactiae. These reporter gene studies also suggest that RogB exerts a positive effect on fbsA expression in S. agalactiae.
doi:10.1128/IAI.71.9.5056-5064.2003
PMCID: PMC187362  PMID: 12933848
14.  Chromosomal Transfers in Mycoplasmas: When Minimal Genomes Go Mobile 
mBio  2014;5(6):e01958-14.
ABSTRACT
Horizontal gene transfer (HGT) is a main driving force of bacterial evolution and innovation. This phenomenon was long thought to be marginal in mycoplasmas, a large group of self-replicating bacteria characterized by minute genomes as a result of successive gene losses during evolution. Recent comparative genomic analyses challenged this paradigm, but the occurrence of chromosomal exchanges had never been formally addressed in mycoplasmas. Here, we demonstrated the conjugal transfer of large chromosomal regions within and among ruminant mycoplasma species, with the incorporation of the incoming DNA occurring by homologous recombination into the recipient chromosome. By combining classical mating experiments with high-throughput next-generation sequencing, we documented the transfer of almost every position of the mycoplasma chromosome. Mycoplasma conjugation relies on the occurrence of an integrative conjugative element (ICE) in at least one parent cell. While ICE propagates horizontally from ICE-positive to ICE-negative cells, chromosomal transfers (CTs) occurred in the opposite direction, from ICE-negative to ICE-positive cells, independently of ICE movement. These findings challenged the classical mechanisms proposed for other bacteria in which conjugative CTs are driven by conjugative elements, bringing into the spotlight a new means for rapid mycoplasma innovation. Overall, they radically change our current views concerning the evolution of mycoplasmas, with particularly far-reaching implications given that over 50 species are human or animal pathogens.
IMPORTANCE
Horizontal gene transfers (HGT) shape bacterial genomes and are key contributors to microbial diversity and innovation. One main mechanism involves conjugation, a process that allows the simultaneous transfer of significant amounts of DNA upon cell-to-cell contact. Recognizing and deciphering conjugal mechanisms are thus essential in understanding the impact of gene flux on bacterial evolution. We addressed this issue in mycoplasmas, the smallest and simplest self-replicating bacteria. In these organisms, HGT was long thought to be marginal. We showed here that nearly every position of the Mycoplasma agalactiae chromosome could be transferred via conjugation, using an unconventional mechanism. The transfer involved DNA blocks containing up to 80 genes that were incorporated into the host chromosome by homologous recombination. These findings radically change our views concerning mycoplasma evolution and adaptation with particularly far-reaching implications given that over 50 species are human or animal pathogens.
doi:10.1128/mBio.01958-14
PMCID: PMC4251992  PMID: 25425234
15.  Mapping Antigenic Sites of an Immunodominant Surface Lipoprotein of Mycoplasma agalactiae, AvgC, with the Use of Synthetic Peptides  
Infection and Immunity  2002;70(1):171-176.
As a first step toward the design of an epitope vaccine to prevent contagious agalactia, the strongly immunogenic 55-kDa protein of Mycoplasma agalactiae was studied and found to correspond to the AvgC protein encoded by the avgC gene. The avg genes of M. agalactiae, which encode four variable surface lipoproteins, display a significant homology to the vsp (variable membrane surface lipoproteins) genes of the bovine pathogen Mycoplasma bovis at their promoter region as well as their N-terminus-encoding regions. Some members of the Vsp family are known to be involved in cytoadhesion to host cells. In order to localize immunogenic peptides in the AvgC antigen, the protein sequence was submitted to epitope prediction analysis, and five sets of overlapping peptides, corresponding to five selected regions, were synthesized by Spot synthesis. Reactive peptides were selected by immunobinding assay with sera from infected sheep. The three most immunogenic epitopes were shown to be surface exposed by immunoprecipitation assays, and one of these was specifically recognized by all tested sera. Our study indicates that selected epitopes of the AvgC lipoprotein may be used to develop a peptide-based vaccine which is effective against M. agalactiae infection.
doi:10.1128/IAI.70.1.171-176.2002
PMCID: PMC127643  PMID: 11748179
16.  Anti-Bacterial Activity of Recombinant Human β-Defensin-3 Secreted in the Milk of Transgenic Goats Produced by Somatic Cell Nuclear Transfer 
PLoS ONE  2013;8(6):e65379.
The present study was conducted to determine whether recombinant human β-defensin-3 (rHBD3) in the milk of transgenic goats has an anti-bacterial activity against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Streptococcus agalactiae (S. agalactiae) that could cause mastitis. A HBD3 mammary-specific expression vector was transfected by electroporation into goat fetal fibroblasts which were used to produce fourteen healthy transgenic goats by somatic cell nuclear transfer. The expression level of rHBD3 in the milk of the six transgenic goats ranged from 98 to 121 µg/ml at 15 days of lactation, and was maintained at 90–111 µg/ml during the following 2 months. Milk samples from transgenic goats showed an obvious inhibitory activity against E. coli, S. aureus and S. agalactiae in vitro. The minimal inhibitory concentrations of rHBD3 in milk against E. coli, S. aureus and S. agalactiae were 9.5–10.5, 21.8–23.0 and 17.3–18.5 µg/mL, respectively, which was similar to those of the HBD3 standard (P>0.05). The in vivo anti-bacterial activities of rHBD3 in milk were examined by intramammary infusion of viable bacterial inoculums. We observed that 9/10 and 8/10 glands of non-transgenic goats infused with S. aureus and E. coli became infected. The mean numbers of viable bacteria went up to 2.9×103 and 95.4×103 CFU/ml at 48 h after infusion, respectively; the mean somatic cell counts (SCC) in infected glands reached up to 260.4×105 and 622.2×105 cells/ml, which were significantly higher than the SCC in uninfected goat glands. In contrast, no bacteria was presented in glands of transgenic goats and PBS-infused controls, and the SSC did not significantly change throughout the period. Moreover, the compositions and protein profiles of milk from transgenic and non-transgenic goats were identical. The present study demonstrated that HBD3 were an effective anti-bacterial protein to enhance the mastitis resistance of dairy animals.
doi:10.1371/journal.pone.0065379
PMCID: PMC3683007  PMID: 23799010
17.  Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation 
Molecular Microbiology  2008;67(6):1196-1210.
Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits antigenic diversity by switching the expression of multiple surface lipoproteins called Vpmas (Variable proteins of M. agalactiae). Although phase variation has been shown to play important roles in many host–pathogen interactions, the biological significance and the mechanism of Vpma oscillations remain largely unclear. Here, we demonstrate that all six Vpma proteins are expressed in the type strain PG2 and all undergo phase variation at an unusually high frequency. Furthermore, targeted gene disruption of the xer1 gene encoding a putative site-specific recombinase adjacent to the vpma locus was accomplished via homologous recombination using a replicon-based vector. Inactivation of xer1 abolished further Vpma switching and the ‘phase-locked’ mutants (PLMs) continued to steadily express only a single Vpma product. Complementation of the wild-type xer1 gene in PLMs restored Vpma phase variation thereby proving that Xer1 is essential for vpma inversions. The study is not only instrumental in enhancing our ability to understand the role of Vpmas in M. agalactiae infections but also provides useful molecular approaches to study potential disease factors in other ‘difficult-to-manipulate’ mycoplasmas.
doi:10.1111/j.1365-2958.2007.06103.x
PMCID: PMC2268961  PMID: 18248580
18.  Comparative genomics and the role of lateral gene transfer in the evolution of bovine adapted Streptococcus agalactiae 
In addition to causing severe invasive infections in humans, Streptococcus agalactiae, or group B Streptococcus (GBS), is also a major cause of bovine mastitis. Here we provide the first genome sequence for S. agalactiae isolated from a cow diagnosed with clinical mastitis (strain FSL S3-026). Comparison to eight S. agalactiae genomes obtained from human disease isolates revealed 183 genes specific to the bovine strain. Subsequent polymerase chain reaction (PCR) screening for the presence/absence of a subset of these loci in additional bovine and human strains revealed strong differentiation between the two groups (Fisher exact test: p < 0.0001). The majority of the bovine strain-specific genes (~85%) clustered tightly into eight genomic islands, suggesting these genes were acquired through lateral gene transfer (LGT). This bovine GBS also contained an unusually high proportion of insertion sequences (4.3% of the total genome), suggesting frequent genomic rearrangement. Comparison to other mastitis-causing species of bacteria provided strong evidence for two cases of interspecies LGT within the shared bovine environment: bovine S. agalactiae with Streptococcus uberis (nisin U operon) and Streptococcus dysgalactiae subsp. dysgalactiae (lactose operon). We also found evidence for LGT, involving the salivaricin operon, between the bovine S. agalactiae strain and either Streptococcus pyogenes or Streptococcus salivarius. Our findings provide insight intomechanismsfacilitatingenvironmentaladaptationandacquisitionofpotential virulence factors, while highlighting both the key role LGT has played in the recent evolution of the bovine S. agalactiae strain, and the importance of LGT among pathogens within a shared environment.
doi:10.1016/j.meegid.2011.04.019
PMCID: PMC3139733  PMID: 21536150
Comparative; Genomics; Bovine; Adapted; Streptococcus agalactiae
19.  Adaptation of Saccharomyces cerevisiae to the Herbicide 2,4-Dichlorophenoxyacetic Acid, Mediated by Msn2p- and Msn4p-Regulated Genes: Important Role of SPI1 
The possible roles of 13 Msn2p- and Msn4p-regulated genes in the adaptation of Saccharomyces cerevisiae to the herbicide 2,4-d-dichlorophenoxyacetic acid (2,4-D) were examined. Single deletion of genes involved in defense against oxidizing agents (CTT1, GRX1, and GRX2/TTR1) or encoding chaperones of the HSP70 family (SSA1, SSA4, and SSE2) showed a slight effect. A more significant role was observed for the heat shock genes HSP78, HSP26, HSP104, HSP12, and HSP42, most of which encode molecular chaperones. However, the SPI1 gene, encoding a member of the glycosylphosphatidylinositol-anchored cell wall protein family, emerged as the major determinant of 2,4-D resistance. SPI1 expression reduced the loss of viability of an unadapted yeast population suddenly exposed to the herbicide, allowing earlier growth resumption. Significantly, yeast adaptation to 2,4-D involves the rapid and transient Msn2p- and Msn4p-mediated activation (fivefold) of SPI1 transcription. SPI1 mRNA levels were reduced to values slightly above those in unstressed cells when the adapted population started duplication in the presence of 2,4-D. Since SPI1 deletion leads to the higher β-1,3-glucanase sensitivity of 2,4-D-stressed cells, it was hypothesized that adaptation may involve an Spi1p-mediated increase in the diffusional restriction of the liposoluble acid form of the herbicide across the cell envelope. Such a cell response would avoid a futile cycle due to acid reentry into the cell counteracting the active export of the anionic form, presumably through an inducible plasma membrane transporter(s). Consistent with this concept, the concentration of 14C-labeled 2,4-D in 2,4-D-energized adapted Δspi1 mutant cells and the consequent intracellular acidification are higher than in wild-type cells.
doi:10.1128/AEM.69.7.4019-4028.2003
PMCID: PMC165130  PMID: 12839777
20.  The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells 
An in-depth proteomic comparison of human-induced pluripotent stem cells, and their parent fibroblast cells, with embryonic stem cells shows that the reprogramming process comprehensively remodels protein expression levels, creating cells that closely resemble natural stem cells.
We present here a large proteomic characterization of human embryonic stem cells, human-induced pluripotent stem cells and their parental fibroblasts cell lines.Overall, 97.8% of the 2683 quantified proteins in four experiments showed no significant differences in abundance between hESC and hiPSC highlighting the high similarity of these pluripotent cell lines.In total, 58 proteins were found significantly differentially expressed between hiPSCs and hESCs. The observed low overlap of these proteins with previous transcriptomic studies suggests that those differences do no reflect a recurrent molecular signature.
Human embryonic stem cells (hESCs) are capable of self-renewal and multi-lineage differentiation. However, the use of hESCs for clinical treatment entails ethical issues as they are derived from human embryos. Recently, reprogramming of somatic cells to an embryonic stem cell-like state, named induced pluripotent stem cells (iPSCs), was achieved through ectopic expression of defined factors. In addition to their clinical potential, hiPSCs represent a unique tool to develop cellular models for human diseases as well. Although current functional assays (e.g., tetraploid complementation) have confirmed the pluripotency of hiPSCs, there might still be significant differences (e.g., differentiation potential) when compared with their natural hESCs counterparts. Consequently, an extensive molecular characterization to address differences and similarities between these two pluripotent cell lines seems to be a prerequisite before any clinical application is conducted. Despite that great efforts, mainly at the genomic levels, have been made to address how similar hESCs and hiPSCs are, the definite answer to this fundamental question is currently still debated. Direct assessment of protein levels has yet to be incorporated into these integrative systems-level analyses. Protein levels are tuned by intricate mechanisms of gene expression regulation and it has recently been documented that mRNA and protein levels poorly correlate in mouse ESCs. Here, we use in-depth quantitative proteomics to gain insights into the differences and similarities in the protein content of two hiPS cell lines, their precursor IMR90 and 4Skin fibroblast cell lines and one hES cell line, providing novel molecular signatures that may assist in filling a gap in the understanding of pluripotency.
To study the degree of similarity, at the protein level, between hiPSCs and hESCs, four MS-based proteomic experiments were designed that use our in-house developed triplex dimethyl labeling chemistry followed by extensive fractionation by strong cation exchange (SCX) chromatography to reduce the sample complexity. High-resolution LC-MS/MS with dedicated fragmentation schemes (i.e., electron transfer dissociation, collision-induced dissociation and higher-energy collision dissociation) was subsequently used to maximize peptide identification rates. A total of 348 LC-MS/MS analyses (including technical and biological replicates) were performed. We confidently identified 1 593 446 peptide spectrum matches (peptide FDR<1%) corresponding to 10 628 unique protein groups (protein FDR∼4%). Using the extracted ion chromatograms, we also estimated the absolute abundance of the proteins within the samples spanning six orders of magnitude. To the best of our knowledge, the coverage obtained in this study represents the largest achieved by any proteomics screen on pluripotent cells.
Most importantly, our results indicate that the reprogramming process remodeled the proteome of both fibroblast cell lines to a profile that closely resembles the pluripotent hESCs proteome: 97.8% of the quantified proteins (2638 proteins in all four experiments) showed nonsignificant changes. Nevertheless, a small fraction of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly regulated between hiPSCs and hESCs. A comparison of the regulated proteins to previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. On the other side, the inclusion of the two parental fibroblast cell lines in our analysis allowed us to study changes in the proteome at both the starting and end points of the reprogramming process. As expected, the vast majority of the proteins (73.4%) showed differential expression between the parental fibroblasts and the reprogrammed pluripotent cells.
To find out if the differences observed in our study were a consequence of transcriptional or translational regulation, we performed paired genome-wide gene expression analyses on the same six samples that were used for the proteomic profiling. Overall, we observed a good correlation between mRNA and protein levels (r∼0.7). These results further authenticated the proteomic measurements and implied a high degree of control at the transcriptional level. Nevertheless, numerous genes were found uncorrelated highlighting the necessity of complementing transcriptomic-based approaches with proteomics.
Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature.
doi:10.1038/msb.2011.84
PMCID: PMC3261715  PMID: 22108792
human embryonic stem cells; human-induced pluripotent stem cells; proteomics; quantitation
21.  Surface Diversity in Mycoplasma agalactiae Is Driven by Site-Specific DNA Inversions within the vpma Multigene Locus 
Journal of Bacteriology  2002;184(21):5987-5998.
The ruminant pathogen Mycoplasma agalactiae possesses a family of abundantly expressed variable surface lipoproteins called Vpmas. Phenotypic switches between Vpma members have previously been correlated with DNA rearrangements within a locus of vpma genes and are proposed to play an important role in disease pathogenesis. In this study, six vpma genes were characterized in the M. agalactiae type strain PG2. All vpma genes clustered within an 8-kb region and shared highly conserved 5′ untranslated regions, lipoprotein signal sequences, and short N-terminal sequences. Analyses of the vpma loci from consecutive clonal isolates showed that vpma DNA rearrangements were site specific and that cleavage and strand exchange occurred within a minimal region of 21 bp located within the 5′ untranslated region of all vpma genes. This process controlled expression of vpma genes by effectively linking the open reading frame (ORF) of a silent gene to a unique active promoter sequence within the locus. An ORF (xer1) immediately adjacent to one end of the vpma locus did not undergo rearrangement and had significant homology to a distinct subset of genes belonging to the λ integrase family of site-specific xer recombinases. It is proposed that xer1 codes for a site-specific recombinase that is not involved in chromosome dimer resolution but rather is responsible for the observed vpma-specific recombination in M. agalactiae.
doi:10.1128/JB.184.21.5987-5998.2002
PMCID: PMC135373  PMID: 12374833
22.  Homologue of Macrophage-Activating Lipoprotein in Mycoplasma gallisepticum Is Not Essential for Growth and Pathogenicity in Tracheal Organ Cultures 
Journal of Bacteriology  2003;185(8):2538-2547.
While the genomes of a number of Mycoplasma species have been fully determined, there has been limited characterization of which genes are essential. The surface protein (p47) identified by monoclonal antibody B3 is the basis for an enzyme-linked immunosorbent assay for serological detection of Mycoplasma gallisepticum infection and appears to be constitutively expressed. Its gene was cloned, and the DNA sequence was determined. Subsequent analysis of the p47 amino acid sequence and searches of DNA databases found homologous gene sequences in the genomes of M. pneumoniae and M. genitalium and identity with a gene family in Ureaplasma urealyticum and genes in M. agalactiae and M. fermentans. The proteins encoded by these genes were found to belong to a family of basic membrane proteins (BMP) that are found in a wide range of bacteria, including a number of pathogens. Several of the BMP family members, including p47, contain selective lipoprotein-associated motifs that are found in macrophage-activating lipoprotein 404 of M. fermentans and lipoprotein P48 of M. agalactiae. The p47 gene was predicted to encode a 59-kDa peptide, but affinity-purified p47 had a molecular mass of approximately 47 kDa, as determined by polyacrylamide gel analysis. Analysis of native and recombinant p47 by mass peptide fingerprinting revealed the absence of the carboxyl end of the protein encoded by the p47 gene in native p47, which would account for the difference seen in the predicted and measured molecular weights and indicated posttranslational cleavage of the lipoprotein at its carboxyl end. A DNA construct containing the p47 gene interrupted by the gene encoding tetracycline resistance was used to transform M. gallisepticum cells. A tetracycline-resistant mycoplasma clone, P2, contained the construct inserted within the genomic p47 gene, with crossovers occurring between 73 bp upstream and 304 bp downstream of the inserted tetracycline resistance gene. The absence of p47 protein in clone P2 was determined by the lack of reactivity with rabbit anti-p47 sera or monoclonal antibody B3 in Western blots of whole-cell proteins. There was no difference between the p47− mutant and wild-type M. gallisepticum in pathogenicity in chicken tracheal organ cultures. Thus, p47, although homologous to genes that occur in many prokaryotes, is not essential for growth in vitro or for attachment and the initial stages of pathogenesis in chickens.
doi:10.1128/JB.185.8.2538-2547.2003
PMCID: PMC152605  PMID: 12670978
23.  Distribution and diversity of mycoplasma plasmids: lessons from cryptic genetic elements 
BMC Microbiology  2012;12:257.
Background
The evolution of mycoplasmas from a common ancestor with Firmicutes has been characterized not only by genome down-sizing but also by horizontal gene transfer between mycoplasma species sharing a common host. The mechanisms of these gene transfers remain unclear because our knowledge of the mycoplasma mobile genetic elements is limited. In particular, only a few plasmids have been described within the Mycoplasma genus.
Results
We have shown that several species of ruminant mycoplasmas carry plasmids that are members of a large family of elements and replicate via a rolling-circle mechanism. All plasmids were isolated from species that either belonged or were closely related to the Mycoplasma mycoides cluster; none was from the Mycoplasma bovis-Mycoplasma agalactiae group. Twenty one plasmids were completely sequenced, named and compared with each other and with the five mycoplasma plasmids previously reported. All plasmids share similar size and genetic organization, and present a mosaic structure. A peculiar case is that of the plasmid pMyBK1 from M. yeatsii; it is larger in size and is predicted to be mobilizable. Its origin of replication and replication protein were identified. In addition, pMyBK1 derivatives were shown to replicate in various species of the M. mycoides cluster, and therefore hold considerable promise for developing gene vectors. The phylogenetic analysis of these plasmids confirms the uniqueness of pMyBK1 and indicates that the other mycoplasma plasmids cluster together, apart from the related replicons found in phytoplasmas and in species of the clade Firmicutes.
Conclusions
Our results unraveled a totally new picture of mycoplasma plasmids. Although they probably play a limited role in the gene exchanges that participate in mycoplasma evolution, they are abundant in some species. Evidence for the occurrence of frequent genetic recombination strongly suggests they are transmitted between species sharing a common host or niche.
doi:10.1186/1471-2180-12-257
PMCID: PMC3541243  PMID: 23145790
Mycoplasma,Plasmid,Replication,Rep protein,Gene transfer,Evolution,Expression vector,Mycoplasma mycoides,Mycoplasma capricolum,Mycoplasma yeatsii
24.  Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry 
Journal of Clinical Microbiology  2013;51(10):3314-3323.
Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of ≥1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing.
doi:10.1128/JCM.01573-13
PMCID: PMC3811644  PMID: 23903545
25.  Mycoplasma agalactiae p40 Gene, a Novel Marker for Diagnosis of Contagious Agalactia in Sheep by Real-Time PCR: Assessment of Analytical Performance and In-House Validation Using Naturally Contaminated Milk Samples▿ † 
Journal of Clinical Microbiology  2008;47(2):445-450.
We evaluated the capacity of the Mycoplasma agalactiae p40 gene as a diagnostic marker for contagious agalactia in sheep by quantitative real-time PCR. The p40 gene encodes an immunodominant adhesin that plays a key role in cytoadhesion of M. agalactiae. The assay was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE), a quantification that is highly linear (R2 > 0.992) and efficient (PCR efficiency, >0.992) over a 6-log dynamic range, down to 10 GE. We evaluated the capacity of the assay to detect Mycoplasma agalactiae in 797 milk samples (373 raw sheep milk samples from refrigerated tanks of different farms and 424 milk samples from individual sheep of a flock positive for M. agalactiae). In parallel, we also tested the samples by using microbiological isolation coupled with microscopy identification and by a PCR method recommended by the World Organization for Animal Health. While our assay was able to detect 57 (15.28%) positive samples of the 373 milk samples from different farms, identification by microbiological isolation coupled with microscopy detected only 36 (9.65%) samples, and the conventional PCR detected 31 (8.31%) samples. These findings showed that our assay based on the p40 gene is more specific and sensitive for the detection of M. agalactiae in actual natural samples and, thus, can be a promising alternative tool for diagnosis and epidemiological studies of M. agalactiae infection.
doi:10.1128/JCM.01442-08
PMCID: PMC2643663  PMID: 19020058

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