- Volume 157, Issue 8, 2011
Volume 157, Issue 8, 2011
- Review
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Molecular typing methods for outbreak detection and surveillance of invasive disease caused by Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae, a review
Invasive disease caused by the encapsulated bacteria Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae remains an important cause of morbidity and mortality worldwide, despite the introduction of successful conjugate polysaccharide vaccines that target disease-associated strains. In addition, resistance, or more accurately reduced susceptibility, to therapeutic antibiotics is spreading in populations of these organisms. There is therefore a continuing requirement for the surveillance of vaccine and non-vaccine antigens and antibiotic susceptibilities among isolates from invasive disease, which is only partially met by conventional methods. This need can be met with molecular and especially nucleotide sequence-based typing methods, which are fully developed in the case of N. meningitidis and which could be more widely deployed in clinical laboratories for S. pneumoniae and H. influenzae.
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- Cell And Molecular Biology Of Microbes
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Characterization of DRA0282 from Deinococcus radiodurans for its role in bacterial resistance to DNA damage
More LessDRA0282, a hypothetical protein, was found in a pool of nucleotide-binding proteins in Deinococcus radiodurans cells recovering from gamma radiation stress. This pool exhibited an unusual inhibition of nuclease activity by ATP. The N terminus of DRA0282 showed similarity to human Ku80 homologues, while the C terminus showed no similarities to known proteins. The recombinant protein required Mn2+ for its interaction with DNA and protected dsDNA from exonuclease III degradation. The binding of the protein to supercoiled DNA with a K d of ~2.93 nM was nearly 20-fold stronger than its binding to ssDNA and nearly 67-fold stronger than its binding to linear dsDNA. Escherichia coli cells expressing DRA0282 showed a RecA-dependent enhancement of UV and gamma radiation tolerance. The ΔdrA0282 mutant of D. radiodurans showed a dose-dependent response to gamma radiation. At 14 kGy, the ΔdrA0282 mutant showed nearly 10-fold less survival, while at this dose both pprA : : catΔdrA0282 and pprA : : cat mutants were nearly 100-fold more sensitive than the wild-type. These results suggested that DRA0282 is a DNA-binding protein with a preference for superhelical DNA, and that it plays a role in bacterial resistance to DNA damage through a pathway in which PprA perhaps plays a dominant role in D. radiodurans.
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NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus
Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter–gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.
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Organization of ribonucleoside diphosphate reductase during multifork chromosome replication in Escherichia coli
More LessRibonucleoside diphosphate reductase (RNR) is located in discrete foci in a number that increases with the overlapping of replication cycles in Escherichia coli. Comparison of the numbers of RNR, DnaX and SeqA protein foci with the number of replication forks at different growth rates reveals that fork : focus ratios augment with increasing growth rates, suggesting a higher cohesion of the three protein foci with increasing number of forks per cell. Quantification of NrdB and SeqA proteins per cell showed: (i) a higher amount of RNR per focus at faster growth rates, which sustains the higher cohesion of RNR foci with higher numbers of forks per cell; and (ii) an equivalent amount of RNR per replication fork, independent of the number of the latter.
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Acyl depsipeptide (ADEP) resistance in Streptomyces
More LessADEP, a molecule of the acyl depsipeptide family, has an antibiotic activity with a unique mode of action. ADEP binding to the ubiquitous protease ClpP alters the structure of the enzyme. Access of protein to the ClpP proteolytic chamber is therefore facilitated and its cohort regulatory ATPases (ClpA, ClpC, ClpX) are not required. The consequent uncontrolled protein degradation in the cell appears to kill the ADEP-treated bacteria. ADEP is produced by Streptomyces hawaiiensis. Most sequenced genomes of Streptomyces have five clpP genes, organized as two distinct bicistronic operons, clpP1clpP2 and clpP3clpP4, and a single clpP5 gene. We investigated whether the different Clp proteases are all sensitive to ADEP. We report that ClpP1 is a target of ADEP whereas ClpP3 is largely insensitive. In wild-type Streptomyces lividans, clpP3clpP4 expression is constitutively repressed and the reason for the maintenance of this operon in Streptomyces has been elusive. ClpP activity is indispensable for survival of actinomycetes; we therefore tested whether the clpP3clpP4 operon, encoding an ADEP-insensitive Clp protease, contributes to a mechanism of ADEP resistance by target substitution. We report that in S. lividans, inactivation of ClpP1ClpP2 production or protease activity is indeed a mode of resistance to ADEP although it is neither the only nor the most frequent mode of resistance. The ABC transporter SclAB (orthologous to the Streptomyces coelicolor multidrug resistance pump SCO4959–SCO4960) is also able to confer ADEP resistance, and analysis of strains with sclAB deletions indicates that there are also other mechanisms of ADEP resistance.
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Identification of the in vitro target of an iron-responsive AraC-like protein from Neisseria meningitidis that is in a regulatory cascade with Fur
More LessIn this study we characterized a genetic locus that is predicted to encode one of the three AraC-like regulators of Neisseria meningitidis, a homologue of MpeR of Neisseria gonorrhoeae which is specific to the pathogenic Neisseria species. Previous microarray studies have suggested that this gene is a member of the Fur regulon. In strain MC58, it is a pseudogene (annotated as two ORFs, NMB1879 and NMB1878) containing a frameshift mutation which we show is common to all strains tested belonging to the ST-32 hypervirulent clonal complex. Using primer extension and S1 nuclease protection assays, we mapped two promoters in the upstream intergenic region: the mpeR promoter and the NMB1880 promoter. The latter promoter drives transcription of the divergent upstream locus, which is predicted to encode a high-affinity iron uptake system. We demonstrated that both promoters are induced during iron limitation and that this regulation is also mediated by the Fur regulator. DNA-binding studies with the purified MpeR protein revealed that it binds to a region directly upstream of the NMB1880 divergent promoter, suggesting a role in its regulation. Mutants of N. meningitidis strains lacking MpeR or overexpressing MpeR showed no significant differences in expression of the P NMB1880 promoter, nor did global transcriptional profiling of an MpeR knockout identify any deregulated genes, suggesting that the MpeR protein is inactive under the conditions used in these experiments. The presence of MpeR in a regulatory cascade downstream of the Fur master iron regulator implicates it as being expressed in the iron-limiting environment of the host, where it may in turn regulate a group of genes, including the divergent iron transport locus, in response to signals important for infection.
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Regulation of d-alanylation of lipoteichoic acid in Streptococcus gordonii
More Lessd-Alanyl esters on lipoteichoic acid (LTA) are involved in adhesion, biofilm formation, resistance to cationic antimicrobial peptides, and immune stimulation. There is evidence that bacteria can modulate the level of d-alanyl esters on LTA in response to challenge, but the mechanism of regulation appears to be different among bacteria. In this study, expression of the dlt operon responsible for d-alanylation of LTA was examined in the commensal bacterium Streptococcus gordonii. dlt expression was assessed using the dlt promoter–lacZ reporter gene assay, LTA d-alanine content measurements and dlt mRNA quantification. The results showed that dlt expression was growth phase-dependent, with the greatest expression at the mid-exponential phase of growth. In contrast to Staphylococcus aureus, dlt expression in Strep. gordonii was not affected by the exogenous addition of Mg2+ or K+. Interestingly, dlt expression was upregulated under acidic conditions or when cells were stressed with polymyxin B, indicating that cell envelope stress may be a signal for dlt expression. In view of these results, mutants defective in the cell envelope stress LiaSR two-component regulatory system were constructed. The liaS and liaR mutants showed an increase in dlt expression over the parent strain at neutral pH. The mutants failed to respond to low pH and polymyxin B stress; dlt expression remained the same in the presence or absence of these stresses. These results suggest that dlt expression in Strep. gordonii is regulated by the LiaSR regulatory system in response to environmental signals such as pH and polymyxin B. The regulation appears to be complex, involving both repression and activation mechanisms.
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Selection of hyperadherent mutants in Pseudomonas putida biofilms
More LessA number of genetic determinants required for bacterial colonization of solid surfaces and biofilm formation have been identified in different micro-organisms. There are fewer accounts of mutations that favour the transition to a sessile mode of life. Here we report the isolation of random transposon Pseudomonas putida KT2440 mutants showing increased biofilm formation, and the detailed characterization of one of them. This mutant exhibits a complex phenotype, including altered colony morphology, increased production of extracellular polymeric substances and enhanced swarming motility, along with the formation of denser and more complex biofilms than the parental strain. Sequence analysis revealed that the pleiotropic phenotype exhibited by the mutant resulted from the accumulation of two mutations: a transposon insertion, which disrupted a predicted outer membrane lipoprotein, and a point mutation in lapG, a gene involved in the turnover of the large adhesin LapA. The contribution of each alteration to the phenotype and the possibility that prolonged sessile growth results in the selection of hyperadherent mutants are discussed.
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The autoregulator receptor homologue AvaR3 plays a regulatory role in antibiotic production, mycelial aggregation and colony development of Streptomyces avermitilis
More LessThe γ-butyrolactone autoregulator receptor has been shown to control secondary metabolism and/or morphological differentiation across many Streptomyces species. Streptomyces avermitilis produces an important anthelmintic agent (avermectin) and two further polyketide antibiotics, filipin and oligomycin. Genomic analysis of S. avermitilis revealed that this micro-organism has the clustered putative autoregulator receptor genes distant from the antibiotic biosynthetic gene clusters. Here, we describe the characterization of avaR3, one of the clustered receptor genes, which encodes a protein containing an extra stretch of amino acid residues that has not been found in the family of autoregulator receptors. Disruption of avaR3 resulted in markedly decreased production of avermectins, with delayed expression of avermectin biosynthetic genes, suggesting that AvaR3 positively controls the avermectin biosynthetic genes. Moreover, the disruption caused increased production of filipin without any changes in the transcriptional profile of the filipin biosynthetic genes, suggesting that filipin production is indirectly controlled by AvaR3. The avaR3 disruptant displayed fragmented growth in liquid culture and conditional morphological defects on solid medium. These findings demonstrated that AvaR3 acts as a global regulator that controls antibiotic production and cell morphology.
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- Environmental And Evolutionary Microbiology
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Evolution of the IncP-7 carbazole-degradative plasmid pCAR1 improves survival of its host Pseudomonas fluorescens Pf0-1 in artificial water microcosms
More LessIn our previous study, Pseudomonas fluorescens Pf0-1L, harbouring the IncP-7 carbazole-degradative plasmid pCAR1 : : rfp, was shown to be undetectable within 5 days post-inoculation in carbazole-contaminated artificial freshwater microcosms containing several plasmid-free bacteria in addition to Pf0-1L(pCAR1 : : rfp). Fourteen days after the inoculation, carbazole degraders become detectable. Here, we revealed that these isolates were not pCAR1 transconjugants, but Pf0-1L(pCAR1 : : rfp) mutants, based on RFLP and BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) analysis. Notably, the mutants displayed more rapid initiation of carbazole degradation than the parent strain Pf0-1L(pCAR1 : : rfp). The mutants were unable to degrade anthranilate due to a 163 bp deletion in the antA gene, which was overcome by their transformation with a wild-type antABC-expressing plasmid. Quantitative RT-PCR analysis indicated that the transcriptional induction of carbazole-, anthranilate- and catechol-degradative genes was comparable in both parent and mutant strains. The deletion mutants became dominant in the artificial water microcosm. The mutation caused anthranilate to accumulate instead of catechol, a toxic compound for the parent strain, and may be beneficial to host survival in artificial microcosms.
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Identification of a novel subgroup of uncultured gammaproteobacterial glycogen-accumulating organisms in enhanced biological phosphorus removal sludge
More LessThe presence of glycogen-accumulating organisms (GAO) has been hypothesized to be a cause of deterioration in enhanced biological phosphorus removal (EBPR) processes due to their abilities to out-compete polyphosphate-accumulating organisms (PAO). Based on 16S rRNA gene sequences, new members of uncultured gammaproteobacterial GAO (GB) were identified from sludge samples of a lab-scale sequencing batch reactor used for EBPR. The new GB formed a phylogenetic lineage (GB8) clearly distinct from the previously reported seven GB subgroups. Because the new GB8 members were not targeted by the known fluorescence in situ hybridization (FISH) oligonucleotide probes, a GB8-specific FISH probe (GB429) and a new FISH probe (GB742) targeting all eight GB subgroups were designed, and the phenotypic properties of the new GB8 members were investigated. FISH and microautoradiography approaches showed that GB429-targeted cells (GB8) were large coccobacilli (2–4 µm in size) with the ability to take up acetate under anaerobic conditions, but unable to accumulate polyphosphate under the subsequent aerobic conditions, consistent with in situ phenotypes of GB. FISH analyses on several sludge samples showed that members of GB8 were commonly detected as the majority of GB in lab- and full-scale EBPR processes. In conclusion, this study showed that members of GB8 could be a subgroup of GB with an important role in EBPR deterioration. Designs of FISH probes which hybridize with broader GB subgroups at different hierarchical levels will contribute to studies of the distributions and ecophysiologies of GB in lab- or full-scale EBPR plants.
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- Genes And Genomes
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Hyphal induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein profile
The ability of Candida albicans to switch from yeast to hyphal growth is essential for its virulence. The walls and especially the covalently attached wall proteins are involved in the primary host–pathogen interactions. Three hyphal induction methods were compared, based on fetal calf serum, the amino sugar N-acetylglucosamine (GlcNAc) and the mammalian cell culture medium Iscove’s modified Dulbecco’s medium (IMDM). GlcNAc and IMDM were preferred, allowing stable hyphal growth over a prolonged period without significant reversion to yeast growth and with high biomass yields. We employed Fourier transform-MS combined with a 15N-metabolically labelled reference culture as internal standard for relative quantification of changes in the wall proteome upon hyphal induction. A total of 21 wall proteins were quantified. Our induction methods triggered a similar response characterized by (i) a category of wall proteins showing strongly increased incorporation levels (Als3, Hwp2, Hyr1, Plb5 and Sod5), (ii) another category with strongly decreased levels (Rhd3, Sod4 and Ywp1) and (iii) a third one enriched for carbohydrate-active enzymes (including Cht2, Crh11, Mp65, Pga4, Phr1, Phr2 and Utr2) and showing only a limited response. This is, to our knowledge, the first systematic, quantitative analysis of the changes in the wall proteome of C. albicans upon hyphal induction. Finally, we propose new wall-protein-derived candidates for vaccine development.
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Diversity and distribution of transcription factors: their partner domains play an important role in regulatory plasticity in bacteria
More LessThe ability of bacteria to deal with diverse environmental changes depends on their repertoire of genes and their ability to regulate their expression. In this process, DNA-binding transcription factors (TFs) have a fundamental role because they affect gene expression positively and/or negatively depending on operator context and ligand-binding status. Here, we show an exhaustive analysis of winged helix–turn–helix domains (wHTHs), a class of DNA-binding TFs. These proteins were identified in high proportions and widely distributed in bacteria, representing around half of the total TFs identified so far. In addition, we evaluated the repertoire of wHTHs in terms of their partner domains (PaDos), identifying a similar trend, as with TFs, i.e. they are abundant and widely distributed in bacteria. Based on the PaDos, we defined three main groups of families: (i) monolithic, those families with little PaDo diversity, such as LysR; (ii) promiscuous, those families with a high PaDo diversity; and (iii) monodomain, with families of small sizes, such as MarR. These findings suggest that PaDos have a very important role in the diversification of regulatory responses in bacteria, probably contributing to their regulatory complexity. Thus, the TFs discriminate over longer regions on the DNA through their diverse DNA-binding domains. On the other hand, the PaDos would allow a great flexibility for transcriptional regulation due to their ability to sense diverse stimuli through a variety of ligand-binding compounds.
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- Microbial Pathogenicity
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Rga is a regulator of adherence and pilus formation in Streptococcus agalactiae
Streptococcus agalactiae is the leading cause of bacterial sepsis and meningitis in neonates and is also the causative agent of several serious infections in immunocompromised adults. S. agalactiae encounters multiple niches during an infection, suggesting that regulatory mechanisms control the expression of specific virulence factors in this bacterium. The present study describes the functional characterization of a gene from S. agalactiae, designated rga, which encodes a protein with significant similarity to members of the RofA-like protein (RALP) family of transcriptional regulators. After deletion of the rga gene in the genome of S. agalactiae, the mutant strain exhibited significantly reduced expression of the genes srr-1 and pilA, which encode a serine-rich repeat surface glycoprotein and a pilus protein, respectively, and moderately increased expression of the fbsA gene, which encodes a fibrinogen-binding protein. Electrophoretic mobility shift assays demonstrated specific DNA binding of purified Rga to the promoter regions of pilA and fbsA, suggesting that Rga directly controls pilA and fbsA. Adherence assays revealed significantly reduced binding of the Δrga mutant to epithelial HEp-2 cells and to immobilized human keratin 4, respectively. In contrast, the adherence of the Δrga mutant to A549 cells and its binding to human fibrinogen was significantly increased. Immunoblot and immunoelectron microscopy revealed that the quantity of pilus structures was significantly reduced in the Δrga mutant compared with the parental strain. The wild-type phenotype could be restored by plasmid-mediated expression of rga, demonstrating that the mutant phenotypes resulted from a loss of Rga function.
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Role of Mycoplasma pneumoniae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mediating interactions with the human extracellular matrix
More LessIn different, phylogenetically unrelated micro-organisms, glycolytic enzymes play a dual role. In the cytosol they are involved in metabolic reactions whereas the surface-localized fraction of the enzymes contributes to adhesion and virulence. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a typical member of this group of multifunctional proteins. In this study, we characterized the GAPDH of Mycoplasma pneumoniae, a common pathogen of the human respiratory mucosa. Full-length GAPDH of M. pneumoniae was successfully expressed and used to produce a polyclonal antiserum. By immunofluorescence, colony blot and ELISA experiments with different fractions of the M. pneumoniae proteins, GAPDH was demonstrated to be present in the cytosol and at even higher concentrations at the surface of mycoplasmas. Nevertheless, antibodies against recombinant GAPDH were not detected in sera of immunized animals or of patients with confirmed M. pneumoniae infection. Recombinant GAPDH bound to different human cell lines in a concentration-dependent manner, and binding was inhibited by specific anti-GAPDH serum. In contrast, this antiserum did not significantly influence the adherence of M. pneumoniae to HeLa cells. When different human extracellular matrix proteins were tested in Western blot assays, GAPDH bound to fibrinogen. The results showed that the GAPDH of M. pneumoniae is a member of the family of cytosol-localized glycolytic enzymes, which also occur at the surface of the bacterium, and mediates interactions with the extracellular matrix proteins of the human host. Thus, the surface-exposed fraction of GAPDH may be a factor that contributes to the successful colonization of the human respiratory tract by M. pneumoniae.
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Phosphorylation of the epidermal growth factor receptor (EGFR) is essential for interleukin-8 release from intestinal epithelial cells in response to challenge with Escherichia coli O157 : H7 flagellin
Enterohaemorrhagic Escherichia coli O157 : H7 is a major foodborne and environmental pathogen responsible for both sporadic cases and outbreaks of food poisoning, which can lead to serious sequelae, such as haemolytic uraemic syndrome. The structural subunit of E. coli O157 : H7 flagella is flagellin, which is both the antigenic determinant of the H7 serotype, an important factor in colonization, and an immunomodulatory protein that has been determined to be a major pro-inflammatory component through the instigation of host cell signalling pathways. Flagellin has highly conserved N- and C-terminal regions that are recognized by the host cell pattern recognition receptor Toll-like receptor (TLR) 5. Activation of this receptor triggers cell signalling cascades, which are known to activate host cell kinases and transcription factors that respond with the production of inflammatory mediators such as the chemokine interleukin-8 (IL-8), although the exact components of this pathway are not yet fully characterized. We demonstrate that E. coli O157 : H7-derived flagellin induces rapid phosphorylation of the epidermal growth factor receptor (EGFR), as an early event in intestinal epithelial cell signalling, and that this is required for the release of the pro-inflammatory cytokine IL-8.
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Melanogenesis in dermatophyte species in vitro and during infection
More LessDermatophytes are keratinophilic fungi that are the most common cause of fungal skin infections worldwide. Melanin has been isolated from several important human fungal pathogens, and the polymeric pigment is now recognized as an important virulence determinant. This study investigated whether dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum gypseum, produce melanin or melanin-like compounds in vitro and during infection. Digestion of the pigmented microconidia and macroconidia of dermatophytes with proteolytic enzymes, denaturant and hot concentrated acid yielded dark particles that retained the size and shape of the original fungal cells. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical signal, consistent with the pigments being a melanin. Immunofluorescence analysis demonstrated reactivity of a melanin-binding mAb with the pigmented conidia and hyphae, as well as the isolate particles. Laccase, an enzyme involved in melanization, was detected in the dermatophytes by an agar plate assay using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Skin scrapings from patients with dermatophytoses contained septate hyphae and arthrospores that were reactive with the melanin-binding mAb. These findings indicate that dermatophytes can produce melanin or melanin-like compounds in vitro and during infection. Based on what is known about the function of melanin as a virulence factor of other pathogenic fungi, this pigment may have a similar role in the pathogenesis of dermatophytic diseases.
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The vapA co-expressed virulence plasmid gene vcgB (orf10) of the intracellular actinomycete Rhodococcus equi
The virulence plasmid of the pathogenic actinomycete Rhodococcus equi is essential for proliferation of this pathogen in macrophages and the development of disease. The pathogenicity island of this plasmid encodes a family of virulence-associated proteins (Vap), one of which (VapA) is a virulence factor. This paper describes the vcgAB operon ( vapA co-expressed gene), located upstream of the vapA operon. Transcription of the vcgAB operon gave rise to transcripts with a half-life similar to those determined for other virulence plasmid genes (1.8 min). Transcription started at a promoter similar to the vapA promoter, and proceeded through an inefficient terminator into the downstream vcgC gene. In addition, vcgC is also transcribed from a promoter downstream of vcgB. The vcgAB and vapA operons were coordinately regulated by temperature and pH in a synergistic manner. The latter parameter only affected transcription at higher growth temperatures, indicating that temperature is the dominant regulatory signal. Transcription of the vcgAB operon increased 10-fold during the late exponential and stationary growth phases. Transcription was also upregulated during the initial hours following phagocytosis by phagocytic cells. In contrast to vcgA and vcgC, the vcgB gene is conserved in the porcine VapB-encoding plasmid, as well as in pathogenic mycobacteria. The coordinated regulation of vcgB and vapA, transcription of vcgB following phagocytosis and conservation of vcgB in pathogenic mycobacteria indicate a role for vcgB and the vcg genes in the virulence of R. equi.
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BgaA acts as an adhesin to mediate attachment of some pneumococcal strains to human epithelial cells
More LessStreptococcus pneumoniae colonization of the respiratory tract is an essential precursor for pneumococcal disease. To colonize efficiently, bacteria must adhere to the epithelial-cell surface. S. pneumoniae possesses surface-associated exoglycosidases that are capable of sequentially deglycosylating human glycans. Two exoglycosidases, neuraminidase (NanA) and β-galactosidase (BgaA), have previously been shown to contribute to S. pneumoniae adherence to human epithelial cells, as deletion of either of these genes results in reduced adherence. It has been suggested that these enzymes may modulate adherence by cleaving sugars to reveal a receptor on host cells. Pretreatment of epithelial cells with exogenous neuraminidase restores the adherence of a nanA mutant, whereas pretreatment with β-galactosidase does not restore the adherence of a bgaA mutant. These data suggest that BgaA may not function to reveal a receptor, and implicate an alternative role for BgaA in adherence. Here we demonstrate that β-galactosidase activity is not required for BgaA-mediated adherence. Addition of recombinant BgaA (rBgaA) to adherence assays and pretreatment of epithelial cells with rBgaA both significantly reduced the level of adherence of the parental strain, but not the BgaA mutant. One possible explanation of these data is that BgaA is acting as an adhesin and that rBgaA is binding to the receptor, preventing bacterial binding. A bead-binding assay demonstrated that BgaA can bind directly to human epithelial cells, supporting the hypothesis that BgaA is an adhesin. Preliminary characterization of the epithelial-cell receptor suggests that it is a glycan in the context of a glycosphingolipid. To further establish the relevance of this adherence mechanism, we demonstrated that BgaA-mediated adherence contributed to adherence of a recent clinical isolate to primary human epithelial cells. Together, these data suggest a novel role for BgaA as an adhesin and suggest that this mechanism could contribute to adherence of at least some pneumococcal strains in vivo.
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Tannerella forsythia invasion in oral epithelial cells requires phosphoinositide 3-kinase activation and clathrin-mediated endocytosis
More LessTannerella forsythia, a Gram-negative anaerobe implicated in periodontitis, has been detected within human buccal epithelial cells and shown to invade oral epithelial cells in vitro. We have previously shown that this bacterium triggers host tyrosine kinase-dependent phosphorylation and actin-dependent cytoskeleton reorganization for invasion. On the bacterial side, the leucine-rich repeat cell-surface BspA protein is important for entry. The present study was undertaken to identify host signalling molecules during T. forsythia entry into human oral and cervical epithelial cells. Specifically, the roles of phosphatidylinositol 3-kinase (PI3K), Rho-family GTPases, cholesterol-rich membrane microdomains and the endocytic protein clathrin were investigated. For this purpose, cell lines were pretreated with chemical inhibitors or small interfering RNAs (siRNAs) that target PI3Ks, Rho GTPases, clathrin and cholesterol (a critical component of ‘lipid rafts’), and the resulting effects on T. forsythia uptake were determined. Our studies revealed that T. forsythia entry is dependent on host PI3K signalling, and that purified BspA protein causes activation of this lipid kinase. Bacterial entry also requires the cooperation of host Rac1 GTPase. Finally, our findings indicate an important role for clathrin and cholesterol-rich lipid microdomains in the internalization process
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Volume 61 (1970)
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Volume 60 (1970)
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Volume 59 (1969)
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Volume 58 (1969)
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Volume 57 (1969)
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Volume 56 (1969)
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Volume 55 (1969)
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Volume 54 (1968)
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Volume 53 (1968)
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Volume 52 (1968)
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Volume 51 (1968)
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Volume 50 (1968)
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Volume 49 (1967)
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Volume 48 (1967)
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Volume 47 (1967)
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Volume 46 (1967)
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Volume 45 (1966)
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Volume 44 (1966)
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Volume 43 (1966)
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Volume 42 (1966)
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Volume 41 (1965)
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Volume 40 (1965)
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Volume 39 (1965)
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Volume 38 (1965)
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Volume 37 (1964)
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Volume 36 (1964)
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Volume 35 (1964)
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Volume 34 (1964)
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Volume 33 (1963)
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Volume 32 (1963)
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Volume 31 (1963)
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Volume 30 (1963)
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Volume 29 (1962)
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Volume 28 (1962)
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Volume 27 (1962)
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Volume 26 (1961)
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Volume 25 (1961)
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Volume 24 (1961)
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Volume 23 (1960)
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Volume 22 (1960)
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Volume 21 (1959)
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Volume 20 (1959)
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Volume 19 (1958)
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Volume 18 (1958)
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Volume 17 (1957)
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Volume 16 (1957)
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Volume 15 (1956)
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Volume 14 (1956)
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Volume 13 (1955)
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Volume 12 (1955)
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Volume 11 (1954)
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Volume 10 (1954)
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Volume 9 (1953)
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Volume 8 (1953)
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Volume 7 (1952)
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Volume 6 (1952)
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Volume 5 (1951)
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Volume 4 (1950)
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Volume 3 (1949)
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Volume 2 (1948)
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Volume 1 (1947)