- Volume 155, Issue 10, 2009
Volume 155, Issue 10, 2009
- Review
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Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis
More LessThe glyoxylate cycle is an anaplerotic pathway of the tricarboxylic acid (TCA) cycle that allows growth on C2 compounds by bypassing the CO2-generating steps of the TCA cycle. The unique enzymes of this route are isocitrate lyase (ICL) and malate synthase (MS). ICL cleaves isocitrate to glyoxylate and succinate, and MS converts glyoxylate and acetyl-CoA to malate. The end products of the bypass can be used for gluconeogenesis and other biosynthetic processes. The glyoxylate cycle occurs in Eukarya, Bacteria and Archaea. Recent studies of ICL- and MS-deficient strains as well as proteomic and transcriptional analyses show that these enzymes are often important in human, animal and plant pathogenesis. These studies have extended our understanding of the metabolic pathways essential for the survival of pathogens inside the host and provide a more complete picture of the physiology of pathogenic micro-organisms. Hopefully, the recent knowledge generated about the role of the glyoxylate cycle in virulence can be used for the development of new vaccines, or specific inhibitors to combat bacterial and fungal diseases.
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- Mini-Review
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Pumping iron: mechanisms for iron uptake by Campylobacter
More LessCampylobacter requires iron for successful colonization of the host. In the last 7 years, a wealth of data has been generated allowing detailed molecular characterization of Campylobacter iron-uptake systems. Several exogenous siderophores have been identified as sources of ferric iron for Campylobacter. Ferri-enterochelin uptake requires both the outer-membrane receptor protein CfrA and the inner-membrane ABC transporter system CeuBCDE. Ferrichrome has been shown to support growth of some Campylobacter jejuni strains and the presence of homologues of Escherichia coli fhuABD genes was proposed; the Cj1658–Cj1663 system appears to be involved in the uptake of ferri-rhodotorulic acid. In addition to siderophores, the importance of host iron sources was highlighted by recent studies demonstrating that C. jejuni can exploit haem compounds and the transferrins using ChuABCDZ and Cj0173c–Cj0178, respectively. An additional putative receptor, Cj0444, present in some, but not all, strains has not yet been characterized. Following diffusion through the outer membrane, inner-membrane transport of ferrous iron can occur via the FeoB protein. While it may be assumed that all systems are not essential, there is growing evidence supporting the need for multiple iron-uptake systems for successful host colonization by Campylobacter. In light of this, comparative molecular characterization of iron systems in all Campylobacter strains is necessary to gain further insight into the pathogenesis of members of this genus.
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- Cell And Molecular Biology Of Microbes
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Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host
The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.
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Presence of the Fps1p aquaglyceroporin channel is essential for Hog1p activation, but suppresses Slt2(Mpk1)p activation, with acetic acid stress of yeast
More LessWhen grown at pH 4.5, Saccharomyces cerevisiae acquires a resistance to inhibitory acetic acid levels (∼0.1 M) by destabilizing Fps1p, the plasma membrane aquaglyceroporin that provides the main route for passive diffusional entry of this acid into the cell. Acetic acid stress transiently activates Hog1p mitogen-activated protein (MAP) kinase, which, in turn, phosphorylates Fps1p in order to target this channel for endocytosis and degradation in the vacuole. This activation of Hog1p is abolished with the loss of Fps1p, but is more sustained when cells express an open Fps1p channel refractory to destabilization. At neutral pH, much higher levels of acetate (∼0.5 M) are needed to inhibit growth. Under such conditions, the loss of Fps1p does not abolish, but merely slows, the activation of Hog1p. Acetate stress also activates the Slt2(Mpk1)p cell integrity MAP kinase, possibly by causing inhibition of glucan synthase activity. In pH 4.5 cultures, this acetate activation of Slt2p is strongly enhanced by the loss of Fps1p and is dependent upon the cell surface sensor Wsc1p. Lack of Fps1p therefore exerts opposing effects on the activation of Hog1p and Slt2p in yeast exposed to acetic acid stress.
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Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa
MexXY, a drug efflux pump in Pseudomonas aeruginosa, confers resistance to aminoglycoside antibiotics. We recently reported that MexZ binds to the promoter region of the mexXY operon. Electrophoretic mobility shift assay (EMSA) using recombinant MexZ and oligonucleotide probes prepared from the intergenic region between mexZ and mexX revealed that MexZ binds to a 20 bp palindromic sequence. Culture of P. aeruginosa in the presence of tetracycline induced higher levels of MexX and MexZ, as measured by immunoblotting and EMSA, than in the absence of antibiotics. When MexZ was expressed by a mexZ expression plasmid, the plasmid-borne MexZ repressed drug-induced MexX production, further confirming that MexZ acts as a repressor of the mexXY operon. PA5471 protein has been reported to be essential for drug-induced MexXY production. Similarly to that report, we observed that plasmid-borne PA5471 induced both MexX and MexZ production in PAO1 cells. Interestingly, interaction between MexZ and PA5471 was observed in a yeast two-hybrid assay. Furthermore, EMSA and in vitro transcription assays revealed that interaction between PA5471 and MexZ reduced MexZ DNA-binding ability, leading to mexXY transcription. These findings contribute to the understanding of the molecular mechanisms underlying the transcriptional regulation of mexZ and mexXY by drug-induced PA5471 expression.
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Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation
More LessStreptococcus mutans in dental biofilms is regularly exposed to cycles of acidic pH during the ingestion of fermentable dietary carbohydrates. The ability of S. mutans to tolerate low pH is crucial for its virulence and pathogenesis in dental caries. To better understand its acid tolerance mechanisms, we performed genome-wide transcriptional analysis of S. mutans in response to an acidic pH signal. The preliminary results showed that adaptation of S. mutans to pH 5.5 induced differential expression of nearly 14 % of the genes in the genome, including 169 upregulated genes and 108 downregulated genes, largely categorized into nine functional groups. One of the most interesting findings was that the genes encoding multiple two-component systems (TCSs), including CiaHR, LevSR, LiaSR, ScnKR, Hk/Rr1037/1038 and ComDE, were upregulated during acid adaptation. Real-time qRT-PCR confirmed the same trend in the expression profiles of these genes at pH 5.5. To determine the roles of these transduction systems in acid adaptation, mutants with a deletion of the histidine-kinase-encoding genes were constructed and assayed for the acid tolerance response (ATR). The results revealed that inactivation of each of these systems resulted in a mutant that was impaired in ATR, since pre-exposure of these mutants to pH 5.5 did not induce the same level of protection against lethal pH levels as the parent did. A competitive fitness assay showed that all the mutants were unable to compete with the parent strain for persistence in dual-strain mixed cultures at acidic pH, although, with the exception of the mutant in liaS, little effect was observed at neutral pH. The evidence from this study suggests that the multiple TCSs are required for S. mutans to orchestrate its signal transduction networks for optimal adaptation to acidic pH.
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Anchoring and length regulation of Porphyromonas gingivalis Mfa1 fimbriae by the downstream gene product Mfa2
Porphyromonas gingivalis, a causative agent of periodontitis, has at least two types of thin, single-stranded fimbriae, termed FimA and Mfa1 (according to the names of major subunits), which can be discriminated by filament length and by the size of their major fimbrilin subunits. FimA fimbriae are long filaments that are easily detached from cells, whereas Mfa1 fimbriae are short filaments that are tightly bound to cells. However, a P. gingivalis ATCC 33277-derived mutant deficient in mfa2, a gene downstream of mfa1, produced long filaments (10 times longer than those of the parent), easily detached from the cell surface, similar to FimA fimbriae. Longer Mfa1 fimbriae contributed to stronger autoaggregation of bacterial cells. Complementation of the mutant with the wild-type mfa2 allele in trans restored the parental phenotype. Mfa2 is present in the outer membrane of P. gingivalis, but does not co-purify with the Mfa1 fimbriae. However, co-immunoprecipitation demonstrated that Mfa2 and Mfa1 are associated with each other in whole P. gingivalis cells. Furthermore, immunogold microscopy, including double labelling, confirmed that Mfa2 was located on the cell surface and likely associated with Mfa1 fimbriae. Mfa2 may therefore play a role as an anchor for the Mfa1 fimbriae and also as a regulator of Mfa1 filament length. Two additional downstream genes (pgn0289 and pgn0290) are co-transcribed with mfa1 (pgn0287) and mfa2 (pgn0288), and proteins derived from pgn0289, pgn0290 and pgn0291 appear to be accessory fimbrial components.
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- Environmental And Evolutionary Microbiology
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Genealogical typing of Neisseria meningitidis
More LessDespite the increasing popularity of multilocus sequence typing (MLST), the most appropriate method for characterizing bacterial variation and facilitating epidemiological investigations remains a matter of debate. Here, we propose that different typing schemes should be compared on the basis of their power to infer clonal relationships and investigate the utility of sequence data for genealogical reconstruction by exploiting new statistical tools and data from 20 housekeeping loci for 93 isolates of the bacterial pathogen Neisseria meningitidis. Our analysis demonstrated that all but one of the hyperinvasive isolates established by multilocus enzyme electrophoresis and MLST were grouped into one of six genealogical lineages, each of which contained substantial variation. Due to the confounding effect of recombination, evolutionary relationships among these lineages remained unclear, even using 20 loci. Analyses of the seven loci in the standard MLST scheme using the same methods reproduced this classification, but were unable to support finer inferences concerning the relationships between the members within each complex.
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Phylogenetic evidence for extensive horizontal gene transfer of type III secretion system genes among enterobacterial plant pathogens
More LessThis study uses sequences from four genes, which are involved in the formation of the type III secretion apparatus, to determine the role of horizontal gene transfer in the evolution of virulence genes for the enterobacterial plant pathogens. Sequences of Erwinia, Brenneria, Pectobacterium, Dickeya and Pantoea were compared (a) with one another, (b) with sequences of enterobacterial animal pathogens, and (c) with sequences of plant pathogenic γ and β proteobacteria, to evaluate probable paths of lateral exchange leading to the current distribution of virulence determinants among these micro-organisms. Phylogenies were reconstructed based on hrcC, hrcR, hrcJ and hrcV gene sequences using parsimony and maximum-likelihood algorithms. Virulence gene phylogenies were also compared with several housekeeping gene loci in order to evaluate patterns of lateral versus vertical acquisition. The resulting phylogenies suggest that multiple horizontal gene transfer events have occurred both within and among the enterobacterial plant pathogens and plant pathogenic γ and β proteobacteria. hrcJ sequences are the most similar, exhibiting anywhere from 2 to 50 % variation at the nucleotide level, with the highest degree of variation present between plant and animal pathogen sequences. hrcV sequences are conserved among plant and animal pathogens at the N terminus. The C-terminal domain is conserved only among the enterobacterial plant pathogens, as are the hrcC and hrcR sequences. Additionally, hrcJ and hrcV sequence phylogenies suggest that at least some type III secretion system virulence genes from enterobacterial plant pathogens are related more closely to those of the genus Pseudomonas, a conclusion neither supported nor refuted by hrcC or hrcR.
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Analysis of the Pseudomonas putida CA-3 proteome during growth on styrene under nitrogen-limiting and non-limiting conditions
More LessPseudomonas putida CA-3 is a styrene-degrading bacterium capable of accumulating medium-chain-length polyhydroxyalkanoate (mclPHA) when exposed to limiting concentrations of a nitrogen source in the growth medium. Using shotgun proteomics we analysed global proteome expression in P. putida CA-3 supplied with styrene as the sole carbon and energy source under N-limiting (condition permissive for mclPHA synthesis) and non-limiting (condition non-permissive for mclPHA accumulation) growth conditions in order to provide insight into the molecular response of P. putida CA-3 to limitation of nitrogen when grown on styrene. A total of 1761 proteins were identified with high confidence and the detected proteins could be assigned to functional groups including styrene degradation, energy, nucleotide metabolism, protein synthesis, transport, stress response and motility. Proteins involved in the upper and lower styrene degradation pathway were expressed throughout the 48 h growth period under both nitrogen limitation and excess. Proteins involved in polyhydroxyalkanoate (PHA) biosynthesis, nitrogen assimilation and amino acid transport, and outer membrane proteins were upregulated under nitrogen limitation. PHA accumulation and biosynthesis were only expressed under nitrogen limitation. Nitrogen assimilation proteins were detected on average at twofold higher amounts under nitrogen limitation. Expression of the branched-chain amino acid ABC transporter was up to 16-fold higher under nitrogen-limiting conditions. Branched chain amino acid uptake by nitrogen-limited cultures was also higher than that by non-limited cultures. Outer membrane lipoproteins were expressed at twofold higher levels under nitrogen limitation. This was confirmed by Western blotting (immunochemical detection) of cells grown under nitrogen limitation. Our study provides the first global description of protein expression changes during growth of any organism on styrene and accumulating mclPHA (nitrogen-limited growth).
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Oceanobacter-related bacteria are important for the degradation of petroleum aliphatic hydrocarbons in the tropical marine environment
More LessPetroleum-hydrocarbon-degrading bacteria were obtained after enrichment on crude oil (as a ‘chocolate mousse’) in a continuous supply of Indonesian seawater amended with nitrogen, phosphorus and iron nutrients. They were related to Alcanivorax and Marinobacter strains, which are ubiquitous petroleum-hydrocarbon-degrading bacteria in marine environments, and to Oceanobacter kriegii (96.4–96.5 % similarities in almost full-length 16S rRNA gene sequences). The Oceanobacter-related bacteria showed high n-alkane-degrading activity, comparable to that of Alcanivorax borkumensis strain SK2. On the other hand, Alcanivorax strains exhibited high activity for branched-alkane degradation and thus could be key bacteria for branched-alkane biodegradation in tropical seas. Oceanobacter-related bacteria became most dominant in microcosms that simulated a crude oil spill event with Indonesian seawater. The dominance was observed in microcosms that were unamended or amended with fertilizer, suggesting that the Oceanobacter-related strains could become dominant in the natural tropical marine environment after an accidental oil spill, and would continue to dominate in the environment after biostimulation. These results suggest that Oceanobacter-related bacteria could be major degraders of petroleum n-alkanes spilt in the tropical sea.
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Occurrence and characterization of peptaibols from Trichoderma citrinoviride, an endophytic fungus of cork oak, using electrospray ionization quadrupole time-of-flight mass spectrometry
A cork oak endophytic strain of Trichoderma citrinoviride, previously selected for its antagonistic potential against various fungal pathogens involved in oak decline, was screened for the production of bioactive secondary metabolites. From liquid culture a mixture of polypeptide antibiotics (peptaibols) belonging to the paracelsin family was isolated and characterized. This peptide mixture was purified by column chromatography and preparative TLC on silica gel, and separated by analytical HPLC. It was analysed by MALDI-TOF MS and nano-ESI-QTOF MS. Tandem mass experiments were performed to determine the amino acid sequences based on the fragmentation pattern of selected parent ions. The mixture comprised 20-residue peptides with C-terminal phenylalaninol and N-terminal acetylation. Twenty-eight amino acid sequences were identified, and amino acid exchanges were located in positions 6, 9, 12 and 17. Among them, seven sequences are new as compared to those reported in the database specifically for peptaibols and in the literature. In addition, we obtained experimental evidence suggesting the existence of non-covalent dimeric forms (homo- and hetero-) of the various peptaibol species. The peptide mixture showed strong antifungal activity toward seven important forest tree pathogens, and it was highly toxic in an Artemia salina (brine shrimp) bioassay. These results emphasize the cryptic role of endophytic fungi as a source of novel bioactive natural products and biocontrol agents.
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- Genes And Genomes
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Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport
In this study, differences at the genetic level of 37 Salmonella Enteritidis strains from five phage types (PTs) were compared using comparative genomic hybridization (CGH) to assess differences between PTs. There were approximately 400 genes that differentiated prevalent (4, 6, 8 and 13a) and sporadic (11) PTs, of which 35 were unique to prevalent PTs, including six plasmid-borne genes, pefA, B, C, D, srgC and rck, and four chromosomal genes encoding putative amino acid transporters. Phenotype array studies also demonstrated that strains from prevalent PTs were less susceptible to urea stress and utilized l-histidine, l-glutamine, l-proline, l-aspartic acid, gly-asn and gly-gln more efficiently than PT11 strains. Complementation of a PT11 strain with the transporter genes from PT4 resulted in a significant increase in utilization of the amino acids and reduced susceptibility to urea stress. In epithelial cell association assays, PT11 strains were less invasive than other prevalent PTs. Most strains from prevalent PTs were better biofilm formers at 37 °C than at 28 °C, whilst the converse was true for PT11 strains. Collectively, the results indicate that genetic and corresponding phenotypic differences exist between strains of the prevalent PTs 4, 6, 8 and 13a and non-prevalent PT11 strains that are likely to provide a selective advantage for strains from the former PTs and could help them to enter the food chain and cause salmonellosis.
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Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum
More LessChorismate mutase (CM) catalyses the rearrangement of chorismate to prephenate and is also the first and the key enzyme that diverges the shikimate pathway to either tryptophan (Trp) or phenylalanine (Phe) and tyrosine (Tyr). Corynebacterium glutamicum is one of the most important amino acid producers for the fermentation industry and has been widely investigated. However, the gene(s) encoding CM has not been experimentally identified in C. glutamicum. In this study, the ncgl0819 gene, which was annotated as ‘conserved hypothetical protein’ in the C. glutamicum genome, was genetically characterized to be essential for growth in minimal medium, and a mutant deleted of ncgl0819 was a Phe and Tyr auxotroph. Genetic cloning and expression of ncgl0819 in Escherichia coli resulted in the formation of a new protein (NCgl0819) having CM activity. It was concluded that ncgl0819 encoded the CM of C. glutamicum (CM0819). CM0819 was demonstrated to be a homodimer and is a new member of the monofunctional CMs of the AroQ structural class. The CM0819 activity was not affected by Phe, Tyr or Trp. Two 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthases (DS0950 and DS2098, formerly NCgl0950 and NCgl2098) had been previously identified from C. glutamicum. CM0819 significantly stimulated DAHP synthase (DS2098) activity. Physical interaction between CM0819 and DS2098 was observed. When CM0819 was present, DS2098 activity was subject to allosteric inhibition by chorismate and prephenate. Conserved hypothetical proteins homologous to CM0819 were identified in all known Corynebacterium genomes, suggesting a universal occurrence of CM0819-like CMs in the genus Corynebacterium.
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- Microbial Pathogenicity
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Enterohaemorrhagic Escherichia coli serogroup O111 inhibits NF-κB-dependent innate responses in a manner independent of a type III secreted OspG orthologue
Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) inject a repertoire of effector proteins into host cells via a type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE). OspG is an effector protein initially identified in Shigella that was shown to inhibit the host innate immune response. In this study, we found ospG homologues in EHEC (mainly of serogroup O111) and in Yersinia enterocolitica. The T3SS encoded by the LEE was able to inject these different OspG homologues into host cells. Infection of HeLa cells with EHEC O111 inhibited the NF-κB-dependent innate immune response via a T3SS-dependent mechanism. However, an EHEC O111 ospG mutant was still able to inhibit NF-κB p65 transfer to the nucleus in infected cells stimulated by tumour necrosis factor α (TNF-α). In addition, no difference in the inflammatory response was observed between wild-type EHEC O111 and the isogenic ospG mutant in the rabbit ligated intestinal loop model. These results suggest that OspG is not the sole effector protein involved in the inactivation of the host innate immune system during EHEC O111 infection.
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The (p)ppGpp synthetase RelA contributes to stress adaptation and virulence in Enterococcus faecalis V583
Guanosine penta- and tetraphosphate [(p)ppGpp] are two unusual nucleotides implied in the bacterial stringent response. In many pathogenic bacteria, mutants unable to synthesize these molecules lose their virulence. In Gram-positive bacteria such as Enterococcus faecalis, the synthesis and degradation of (p)ppGpp mainly depend on the activity of a bifunctional enzyme, encoded by the relA gene. By analysing ΔrelA and ΔrelQ (which encodes a protein harbouring a ppGpp synthetase activity) deletion mutants, we showed that RelA is by far the main system leading to (p)ppGpp production under our experimental conditions, and during the development of a stringent response induced by mupirocin. We also constructed a mutant (ΔrelAsp) in which a small part of the relA gene (about 0.7 kbp) encoding the carboxy-terminal domain of the RelA protein was deleted. Both relA mutants were more resistant than the wild-type strain to 0.3 % bile salts, 25 % ethanol and acid (pH 2.3) challenges. Interestingly, the ΔrelAsp mutant grew better than the two other strains in the presence of 1 mM H2O2, but did not display increased tolerance when subjected to lethal doses of H2O2 (45 mM). By contrast, the ΔrelA mutant was highly sensitive to 45 mM H2O2 and displayed reduced growth in a medium containing 1 M NaCl. The two mutants also displayed contrasting virulence phenotypes towards larvae of the Greater Wax Moth infection model Galleria mellonella. Indeed, although the ΔrelA mutant did not display any phenotype, the ΔrelAsp mutant was more virulent than the wild-type strain. This virulent phenotype should stem from its increased ability to proliferate under oxidative environments.
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Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains
More LessPorphyromonas gingivalis activates protease-activated receptors (PARs) on oral keratinocytes, resulting in downstream signalling for an innate immune response. Activation depends on P. gingivalis gingipains, but could be confounded by lipopolysaccharide signalling through Toll-like receptors. We therefore hypothesized that P. gingivalis cleaves oral keratinocyte PARs in an Arg- (Rgp) or Lys- (Kgp) gingipain-specific manner to upregulate pro-inflammatory cytokines. Immortalized human oral keratinocytes (TERT-2) were incubated with wild-type P. gingivalis (ATCC 33277) or strains from a panel of isogenic gingipain deletion mutants: Kgp-deficient (KDP 129); Rgp-deficient (KDP 133); or Kgp- and Rgp-deficient (KDP 136). After incubation with P. gingivalis, keratinocytes were probed with specific antibodies against the N-terminus of PAR-1 and PAR-2. Using flow cytometry and immunofluorescence, receptor cleavage was marked by loss of specific antibody binding to the respective PARs. TERT-2 cells constitutively expressed high levels of PAR-1 and PAR-2, and lower levels of PAR-3. P. gingivalis ATCC 33277 cleaved PAR-1 and PAR-2 in a dose-dependent manner, while the receptors were unaffected by the protease-negative double mutant (KDP 136) at all m.o.i. tested. The single Kgp-negative mutant preferentially cleaved PAR-1, whereas the Rgp-negative mutant cleaved PAR-2. Wild-type or Kgp-negative mutant cleavage of PAR-1 upregulated expression of IL-1α, IL-1β, IL-6 and TNF-α; the Rgp-negative mutant did not modulate these cytokines. Selective cleavage of PAR-1 on oral epithelial cells by P. gingivalis Rgp therefore upregulates expression of pro-inflammatory cytokines.
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Deletion of Braun lipoprotein gene (lpp) and curing of plasmid pPCP1 dramatically alter the virulence of Yersinia pestis CO92 in a mouse model of pneumonic plague
Deletion of the murein (Braun) lipoprotein gene, lpp, attenuates the Yersinia pestis CO92 strain in mouse models of bubonic and pneumonic plague. In this report, we characterized the virulence of strains from which the plasminogen activating protease (pla)-encoding pPCP1 plasmid was cured from either the wild-type (WT) or the Δlpp mutant strain of Y. pestis CO92 in the mouse model of pneumonic infection. We noted a significantly increased survival rate in mice infected with the Y. pestis pPCP−/Δlpp mutant strain up to a dose of 5000 LD50. Additionally, mice challenged with the pPCP − /Δlpp strain had substantially less tissue injury and a strong decrease in the levels of most cytokines and chemokines in tissue homogenates and sera when compared with the WT-infected group. Importantly, the Y. pestis pPCP − /Δlpp mutant strain was detectable in high numbers in the livers and spleens of some of the infected mice. In the lungs of pPCP − /Δlpp mutant-challenged animals, however, bacterial numbers dropped at 48 h after infection when compared with tissue homogenates from 1 h post-infection. Similarly, we noted that this mutant was unable to survive within murine macrophages in an in vitro assay, whereas survivability of the pPCP− mutant within the macrophage environment was similar to that of the WT. Taken together, our data indicated that a significant and possibly synergistic attenuation in bacterial virulence occurred in a mouse model of pneumonic plague when both the lpp gene and the virulence plasmid pPCP1 encoding the pla gene were deleted from Y. pestis.
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The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a
The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded WzzpHS-2 proteins, respectively. In this study, genes wzzB, wzz pHS-2 and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz pHS-2 revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and WzzpHS-2 proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.
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Small heat-shock protein HspL is induced by VirB protein(s) and promotes VirB/D4-mediated DNA transfer in Agrobacterium tumefaciens
Agrobacterium tumefaciens is a Gram-negative plant-pathogenic bacterium that causes crown gall disease by transferring and integrating its transferred DNA (T-DNA) into the host genome. We characterized the chromosomally encoded alpha-crystallin-type small heat-shock protein (α-Hsp) HspL, which was induced by the virulence (vir) gene inducer acetosyringone (AS). The transcription of hspL but not three other α-Hsp genes (hspC, hspAT1, hspAT2) was upregulated by AS. Further expression analysis in various vir mutants suggested that AS-induced hspL transcription is not directly activated by the VirG response regulator but rather depends on the expression of VirG-activated virB genes encoding components of the type IV secretion system (T4SS). Among the 11 virB genes encoded by the virB operon, HspL protein levels were reduced in strains with deletions of virB6, virB8 or virB11. VirB protein accumulation but not virB transcription levels were reduced in an hspL deletion mutant early after AS induction, implying that HspL may affect the stability of individual VirB proteins or of the T4S complex directly or indirectly. Tumorigenesis efficiency and the VirB/D4-mediated conjugal transfer of an IncQ plasmid RSF1010 derivative between A. tumefaciens strains were reduced in the absence of HspL. In conclusion, increased HspL abundance is triggered in response to certain VirB protein(s) and plays a role in optimal VirB protein accumulation, VirB/D4-mediated DNA transfer and tumorigenesis.
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Cytotoxicity of Brucella smooth strains for macrophages is mediated by increased secretion of the type IV secretion system
Some Brucella rough mutants cause cytotoxicity that resembles oncosis and necrosis in macrophages. This cytotoxicity requires the type IV secretion system (T4SS). In rough mutants, the cell-surface O antigen is shortened and the T4SS structure is thus exposed on the surface. Cytotoxicity effector proteins can therefore be more easily secreted. This enhanced secretion of effector proteins might cause the increased levels of cytotoxicity observed. However, whether this cytotoxicity is unique to the rough mutant and is mediated by overexpression of the T4SS has not been definitively determined. To test this, in the present study, a virB inactivation mutant (BMΔvirB) and an overexpression strain (BM-VIR) of a smooth Brucella melitensis strain (BM) were constructed and their cytotoxicity for macrophages and intracellular survival capability were analysed and compared. Cytotoxicity was detected in macrophages infected with higher concentrations of strains BM or BM-VIR, but not in those infected with BMΔvirB. The quorum sensing signal molecule N-dodecanoyl-dl-homoserine lactone (C12-HSL), a molecule that can inhibit expression of virB, inhibited the cytotoxicity of BM and BM-VIR, but not of BMΔvirB. These results indicated that overexpression of virB is responsible for Brucella cytotoxicity in macrophages. Transcription analysis showed that virB is regulated in a cell-density-dependent manner both in in vitro culture and during macrophage infection. When compared with BM, BM-VIR showed a reduced survival capacity in macrophages and mice, but both strains demonstrated similar resistance to in vitro stress conditions designed to simulate intracellular environments. Taken together, the cytotoxicity of Brucella for macrophages is probably mediated by increased secretion of effector proteins that results from overexpression of virB or an increase in the number of bacterial cells. The observation that both inactivation and overexpression of virB are detrimental for Brucella intracellular survival also indicated that the expression of virB is tightly regulated in a cell-density-dependent manner.
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The enzyme phosphoglucomutase (Pgm) is required by Salmonella enterica serovar Typhimurium for O-antigen production, resistance to antimicrobial peptides and in vivo fitness
More LessThe enzyme phosphoglucomutase (Pgm) catalyses the interconversion of glucose 1-phosphate and glucose 6-phosphate and contributes to glycolysis and the generation of sugar nucleotides for biosynthesis. To assess the role of this enzyme in the biology of the pathogen Salmonella enterica serovar Typhimurium we have characterized a pgm deletion mutant in strain SL1344. Compared to SL1344, SL1344 pgm had impaired growth in vitro, was deficient in the ability to utilize galactose as a carbon source and displayed reduced O-antigen polymer length. The mutant was also more susceptible to antimicrobial peptides and showed decreased fitness in the mouse typhoid model. The in vivo phenotype of SL1344 pgm indicated a role for pgm in the early stages of infection, most likely through deficient O-antigen production. Although pgm mutants in other pathogens have potential as live attenuated vaccine strains, SL1344 pgm was not sufficiently attenuated for such use.
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A peroxiredoxin from Mycoplasma hyopneumoniae with a possible role in H2O2 detoxification
More LessMycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, which affects pig farms worldwide, causing heavy economic losses. In the infection process, this bacterium is exposed to reactive oxygen species (ROS) from its own metabolism or generated by the host as one of the strategies used to neutralize the pathogen. Although the presence of classical antioxidant enzymes would be expected in M. hyopneumoniae, important genes directly related to protection against ROS, such as superoxide dismutase, catalases and glutathione peroxidase, have not been identified by sequence homology in the genome sequence annotation. Among the few identified M. hyopneumoniae genes coding for proteins possibly involved with suppression of ROS-mediated damage, one (tpx) coding for a peroxiredoxin (MhPrx) has been recognized. The sequence and phylogenetic analyses perfomed in this study indicate that MhPrx is closely related to the atypical 2-Cys peroxiredoxin subfamily, although it has only one cysteine in its sequence. The MhPrx coding DNA sequence was cloned and expressed in Escherichia coli to produce a recombinant MhPrx (rMhPrx), which was purified and used to immunize mice and produce an anti-MhPrx polyclonal antiserum. Probing of M. hyopneumoniae extracts with this antiserum demonstrated that MhPrx is expressed in all three tested strains (J, 7422 and 7448). Cross-linking assays and size-exclusion chromatography indicate that rMhPrx forms dimers, as has been established for atypical 2-Cys peroxiredoxins. Furthermore, a metal-catalysed oxidation system was used to assay the activity of rMhPrx, showing that it can protect DNA from ROS-mediated damage and may play an essential role during infection.
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The cell wall galactomannan antigen from Malassezia furfur and Malassezia pachydermatis contains β-1,6-linked linear galactofuranosyl residues and its detection has diagnostic potential
More LessLipophilic yeasts of the genus Malassezia are associated with several skin diseases, such as pityriasis versicolor, Malassezia folliculitis, seborrhoeic dermatitis and atopic dermatitis, and are also increasingly associated with catheter-related fungaemia. The cell wall components of pathogenic micro-organisms behave as an antigen and/or ligand of the innate immune response. Live cells of Malassezia furfur and Malassezia pachydermatis did not react with an anti-α-1,2-mannoside antibody. However, they showed a strong hydrophobicity and reactivity with an anti-β-1,3-glucan antibody compared to those of C. albicans. The cell wall polysaccharides of M. furfur and M. pachydermatis were isolated and their structures analysed by 1H and 13C NMR experiments. Both polysaccharides were shown to be β-1,6-linked linear galactofuranosyl polymers with a small amount of mannan. The presence of galactomannan on cells of Malassezia species has not been described previously. The galactomannan did not react with an anti-Aspergillus fumigatus monoclonal antibody which has specificity for β-1,5-linked galactofuranosyl residues. An anti-M. furfur antibody strongly reacted with the galactomannans of M. furfur and M. pachydermatis, but did not react with the galactomannans of Trichophyton rubrum, A. fumigatus or Fonsecaea pedrosoi. The characteristics of the anti-M. furfur antibody suggest that there is potential for diagnosis of Malassezia infections by antigen detection.
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- Physiology And Biochemistry
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Characterization of the Aspergillus fumigatus phosphomannose isomerase Pmi1 and its impact on cell wall synthesis and morphogenesis
More LessPhosphomannose isomerase (PMI) is an enzyme catalysing the interconversion of mannose 6-phosphate (Man-6-P) and fructose 6-phosphate (Fru-6-P). The reaction catalysed by PMI is the first committed step in the synthesis of mannose-containing sugar chains and provides a link between glucose metabolism and mannosylation. In this study, the pmi1 gene was identified to encode PMI in the human fungal pathogen Aspergillus fumigatus. Characterization of A. fumigatus Pmi1 expressed in Escherichia coli revealed that this PMI mainly catalysed the conversion of Fru-6-P to Man-6-P and that its binding affinity for Man-6-P was similar to that of yeast PMIs, but different to those of PMIs from bacteria or animals. Loss of pmi1 was lethal unless Man was provided in the growth medium. However, a Δpmi1 mutant cell showed a significantly reduced growth rate at a high concentration of Man. Biochemical analysis revealed that both inadequate and replete Man led to an accumulation of intracellular Man-6-P and a reduction in the amount of α-glucan in the cell wall. Uncoupling of the link between energy production and glycosylation by deletion of the pmi1 gene led to phenotypes such as defects in cell wall integrity, abnormal morphology and reduced conidiation. Our results reveal that PMI activity is essential for viability and plays a central regulatory role in both cell wall synthesis and energy production in A. fumigatus.
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The effect of environmental conditions on extracellular protease activity in controlled fermentations of Aspergillus niger
More LessProteolytic degradation by host proteases is one of the key issues in the application of filamentous fungi for non-fungal protein production. In this study the influence of several environmental factors on the production of extracellular proteases of Aspergillus niger was investigated systematically in controlled batch cultures. Of all factors investigated in a series of initial screening experiments, culture pH and nitrogen concentration in particular strongly affected extracellular protease activities. For instance, at a culture pH of 4, protease activity was higher than at pH 5, and protease activity increased with increasing concentrations of ammonium as nitrogen source. Interestingly, an interdependence was observed for several of the factors studied. These possible interaction effects were investigated further using a full factorial experimental design. Amongst others, the results showed a clear interaction effect between nitrogen source and nitrogen concentration. Based on the observed interactions, the selection of environmental factors to reduce protease activity is not straightforward, as unexpected antagonistic or synergistic effects occur. Furthermore, not only were the effects of the process parameters on maximum protease activity investigated, but five other protease-related phenotypes were studied as well, such as maximum specific protease activity and maximum protease productivity. There were significant differences in the effect of the environmental parameters on the various protease-related phenotypes. For instance, pH significantly affected final levels of protease activity, but not protease productivity. The results obtained in this study are important for the optimization of A. niger for protein production.
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Nitric oxide mediates the fungal-elicitor-enhanced biosynthesis of antioxidant polyphenols in submerged cultures of Inonotus obliquus
More LessA fungal elicitor prepared from the cell debris of the plant-pathogenic ascomycete Alternaria alternata induces multiple responses by Inonotus obliquus cells, including an increase in generation of nitric oxide (NO), activity of phenylalanine ammonia lyase (PAL) and accumulation of total mycelial phenolic compounds (TMP), but does not trigger production of oxylipins or jasmonic acid (JA). The role of NO in TMP production was investigated via the effects of the NO-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPITO) and the nitric oxide synthase (NOS) inhibitor aminoguanidine (AG). TMP profiles were assayed using 1H NMR spectroscopy combining multivariate pattern recognition strategies. Pretreatment of I. obliquus mycelia with cPITO or AG suppressed not only elicitor-enhanced NO generation and PAL activity, but also the elicitor-induced increase in TMP production. This TMP reduction by either a NO scavenger or a NOS inhibitor was reversed by exogenous addition of either a NO donor, sodium nitroprusside, or JA separately. NMR-based metabonomic analysis of TMP profiles showed that the induced TMP were hispidin analogues including inoscavins, phelligridins, davallialactone and methyldavallialactone, which possess high antioxidant activities. Thus, NO mediates an elicitor-induced increase in production of antioxidant polyphenols in I. obliquus via a signalling pathway independent of oxylipins or JA, a mechanism which differs from those in some higher plants.
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Truncation in the core oligosaccharide of lipopolysaccharide affects flagella-mediated motility in Pseudomonas aeruginosa PAO1 via modulation of cell surface attachment
More LessIn many Gram-negative bacterial species, rough strains producing truncated lipopolysaccharide (LPS) generally exhibit defects in motility compared with smooth strains. However, the role that LPS plays in bacterial motility is not well understood. The goal of this study was to examine the relationship between LPS defects and motility of Pseudomonas aeruginosa. P. aeruginosa wild-type strain PAO1 and three isogenic mutants with defects in the rmlC, migA and wapR genes and producing truncated core oligosaccharide were investigated in terms of motility, attachment to glass and flagella expression. Compared with the wild-type, the three mutants showed significant retardation in both swarming motility on 0.5 % soft-agar plates and swimming motility on 0.3 % soft-agar plates. Moreover, attachment to abiotic surfaces was observed to be stronger in these mutants. The assembly of flagella appeared to be intact in these strains and the ability of individual cells to swim was unaffected. Flagellin proteins prepared from mutants rmlC and rmd, defective in the production of TDP-l-rhamnose and GDP-d-rhamnose, respectively, were compared and a change in molecular mass was observed only in the rmlC mutant. These data indicated that l-rhamnose, and not its enantiomer, d-rhamnose, is incorporated into the flagellin glycan of P. aeruginosa PAO1. The nucleotide-activated sugar precursor TDP-l-rhamnose is therefore shared between LPS biosynthesis and flagellin glycosylation in P. aeruginosa PAO1. Our results suggest that although biochemical precursors are shared by LPS and flagellin glycan biosynthesis, LPS truncations probably alter flagella-mediated motility in P. aeruginosa by modulating cell-surface attachment but not flagella synthesis.
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Conditioning of the membrane fatty acid profile of Escherichia coli during periodic temperature cycling
More LessThe membrane fatty acid composition of Escherichia coli becomes conditioned during periodic temperature cycling between 37 and 8 °C. After several cycles of temperature change, the bacteria become locked into a low-temperature physiology. Even after a prolonged incubation at high temperature the membrane fatty acid composition of conditioned cells was similar to that of cold-stressed cells.
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The protease CspB is essential for initiation of cortex hydrolysis and dipicolinic acid (DPA) release during germination of spores of Clostridium perfringens type A food poisoning isolates
More LessThe genome of the Clostridium perfringens food poisoning isolate SM101 encodes a subtilisin-like protease, CspB, upstream of the sleC gene encoding the enzyme essential for degradation of the peptidoglycan cortex during spore germination. SleC is an inactive pro-SleC in dormant spores that is converted to active SleC during spore germination and Csp proteases convert pro-SleC to the active enzyme in vitro. In this work, the germination and viability of spores of a cspB deletion mutant of strain SM101, as well as cspB expression, were studied. The cspB gene was expressed only during sporulation, and only in the mother cell compartment. cspB spores were unable to germinate significantly with either a rich nutrient medium, KCl, or a 1 : 1 chelate of Ca2+ and dipicolinic acid (DPA); the viability of these spores was ∼104-fold lower than that of wild-type spores, although cspB and wild-type spores had similar viability on plates containing lysozyme, and cspB spores could not process inactive pro-SleC into active SleC during spore germination. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis, and germination and viability defects in these spores were complemented by an ectopic cspB. These results indicate that Csp proteases are essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. However, Csp proteases likely play another role in spore germination, since cspB spores did not release DPA upon exposure to germinants, while sleC spores have been shown previously to release DPA, albeit slowly, upon exposure to germinants.
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