- Volume 157, Issue 12, 2011
Volume 157, Issue 12, 2011
- Microbial Pathogenicity
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The role of Klebsiella pneumoniae rmpA in capsular polysaccharide synthesis and virulence revisited
More LessKlebsiella pneumoniae community-acquired pyogenic liver abscess (PLA) is an emerging infectious disease. The rmpA gene (for regulator of mucoid phenotype A) has been reported to be associated with PLA in prevalence studies. NTUH-K2044, a K1 PLA isolate, carries three rmpA/A2 genes: two large-plasmid-carried genes (p-rmpA and p-rmpA2) and one chromosomal gene (c-rmpA). In this study, we re-examined the role of rmpA/A2 in PLA pathogenesis to clarify the relationship of rmpA/A2 and capsular serotype to virulence. Using isogenic gene deletion strains and complemented strains of NTUH-K2044, we demonstrated that only p-rmpA enhanced expression of capsular polysaccharide synthesis (cps) genes and capsule production. Nevertheless, the lethal dose and in vivo competitive index indicated that p-rmpA does not promote virulence in mice. The prevalence of these three rmpA/A2 and capsular types in 206 strains was investigated. This revealed a correlation of rmpA/A2 with six PLA-related capsular types (K1, K2, K5, K54, K57 and KN1). However, the correlation of rmpA/A2 with K1 strains from the West was less obvious than with the strains from Asia (17/22 vs 39/39, P = 0.0019). Among the three rmpA/A2 genes, p-rmpA was the most prevalent. Due to the strong correlation with PLA-related capsular types, p-rmpA could serve as a surrogate marker for PLA. We found an association of p-rmpA with three widely spaced loci in a large plasmid (30/32). Therefore, rmpA could be co-inherited together with virulence genes carried by this plasmid.
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CcpA coordinates central metabolism and biofilm formation in Staphylococcus epidermidis
Staphylococcus epidermidis is an opportunistic bacterium whose infections often involve the formation of a biofilm on implanted biomaterials. In S. epidermidis, the exopolysaccharide facilitating bacterial adherence in a biofilm is polysaccharide intercellular adhesin (PIA), whose synthesis requires the enzymes encoded within the intercellular adhesin operon (icaADBC). In vitro, the formation of S. epidermidis biofilms is enhanced by conditions that repress tricarboxylic acid (TCA) cycle activity, such as growth in a medium containing glucose. In many Gram-positive bacteria, repression of TCA cycle genes in response to glucose is accomplished by catabolite control protein A (CcpA). CcpA is a member of the GalR–LacI repressor family that mediates carbon catabolite repression, leading us to hypothesize that catabolite control of S. epidermidis biofilm formation is indirectly regulated by CcpA-dependent repression of the TCA cycle. To test this hypothesis, ccpA deletion mutants were constructed in strain 1457 and 1457-acnA and the effects on TCA cycle activity, biofilm formation and virulence were assessed. As anticipated, deletion of ccpA derepressed TCA cycle activity and inhibited biofilm formation; however, ccpA deletion had only a modest effect on icaADBC transcription. Surprisingly, deletion of ccpA in strain 1457-acnA, a strain whose TCA cycle is inactive and where icaADBC transcription is derepressed, strongly inhibited icaADBC transcription. These observations demonstrate that CcpA is a positive effector of biofilm formation and icaADBC transcription and a repressor of TCA cycle activity.
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Pyruvate : ferredoxin oxidoreductase (PFO) is a surface-associated cell-binding protein in Trichomonas vaginalis and is involved in trichomonal adherence to host cells
The Trichomonas vaginalis 120 kDa protein adhesin (AP120) is induced under iron-rich conditions and has sequence homology with pyruvate : ferredoxin oxidoreductase A (PFO A), a hydrogenosomal enzyme that is absent in humans. This homology raises the possibility that, like AP120, PFO might be localized to the parasite surface and participate in cytoadherence. Here, the cellular localization and function of PFO that was expressed under various iron concentrations was investigated using a polyclonal antibody generated against the 50 kDa recombinant C-terminal region of PFO A (anti-PFO50). In Western blot assays, this antibody recognized a 120 kDa protein band in total protein extracts, and proteins with affinity to the surface of HeLa cells from parasites grown under iron-rich conditions. In addition to localization that is typical of hydrogenosomal proteins, PFOs that were expressed under iron-rich conditions were found to localize at the surface. This localization was demonstrated using immunofluorescence and co-localization assays, as well as immunogold transmission electron microscopy. In addition to describing its enzyme activity, we describe a novel function in trichomonal host interaction for the PFO localized on the parasite surface. The anti-PFO50 antibody reduced the levels of T. vaginalis adherence to HeLa cell monolayers in a concentration-dependent manner. Thus, T. vaginalis PFO is an example of a surface-associated cell-binding protein that lacks enzyme activity and that is involved in cytoadherence. Additionally, PFO behaves like AP120 in parasites grown under iron-rich conditions. Therefore, these data suggest that AP120 and PFO A are encoded by the same gene, namely pfo a.
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The biochemical properties of the Francisella pathogenicity island (FPI)-encoded proteins IglA, IglB, IglC, PdpB and DotU suggest roles in type VI secretion
The Francisella pathogenicity island (FPI) encodes proteins thought to compose a type VI secretion system (T6SS) that is required for the intracellular growth of Francisella novicida. In this work we used deletion mutagenesis and genetic complementation to determine that the intracellular growth of F. novicida was dependent on 14 of the 18 genes in the FPI. The products of the iglABCD operon were localized by the biochemical fractionation of F. novicida, and Francisella tularensis LVS. Sucrose gradient separation of water-insoluble material showed that the FPI-encoded proteins IglA, IglB and IglC were found in multiple fractions, especially in a fraction that did not correspond to a known membrane fraction. We interpreted these data to suggest that IglA, IglB and IglC are part of a macromolecular structure. Analysis of published structural data suggested that IglC is an analogue of Hcp, which is thought to form long nano-tubes. Thus the fractionation properties of IglA, IglB and IglC are consistent with the current model of the T6SS apparatus, which supposes that IglA and IglB homologues form an outer tube structure that surrounds an inner tube composed of Hcp (IglC) subunits. Fractionation of F. novicida expressing FLAG-tagged DotU (IcmH homologue) and PdpB (IcmF homologue) showed that these proteins localize to the inner membrane. Deletion of dotU led to the cleavage of PdpB, suggesting an interaction of these two proteins that is consistent with results obtained with other T6SSs. Our results may provide a mechanistic basis for many of the studies that have examined the virulence properties of Francisella mutants in FPI genes, namely that the observed phenotypes of the mutants are the result of the disruption of the FPI-encoded T6SS structure.
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Purification and characterization of a secretory lipolytic enzyme, MgLIP2, from Malassezia globosa
More LessMalassezia globosa is a lipid-dependent yeast that is found on the human skin and is associated with various skin disorders, including dandruff and seborrhoeic dermatitis (SD). Despite its important role in skin diseases, the molecular basis for its pathogenicity is poorly understood. The current hypothesis is that dandruff and SD are linked to fatty acid metabolism and secretory lipolytic enzymes, which hydrolyse sebaceous lipids and release irritating free fatty acids. A previous genomic analysis of M. globosa identified a family of 13 homologous genes predicted to encode secreted lipases. We have also reported that M. globosa had significantly higher extracellular lipase activity compared with other species. To identify the major secretory lipases of this yeast during its growth, we successfully purified and characterized an extracellular lipase MgLIP2. Based on MALDI-TOF MS, the peptide mass fingerprint of a tryptically digested protein MgLIP2 corresponded to ORF MGL_4054 of M. globosa. This lipase showed high esterase activity against 4-nitrophenyl palmitate and 1-naphthyl palmitate but not 1-naphthyl acetate. This enzyme had optimal activity at 30 °C and pH 5.0. Furthermore, the activity significantly increased in the presence of Triton X-100 and was partially inhibited by PMSF but was unaffected by univalent and divalent metal ions.
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- Physiology and Biochemistry
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Dynamic regulation of mitochondrial respiratory chain efficiency in Saccharomyces cerevisiae
To adapt to changes in the environment, cells have to dynamically alter their phenotype in response to, for instance, temperature and oxygen availability. Interestingly, mitochondrial function in Saccharomyces cerevisiae is inherently temperature sensitive; above 37 °C, yeast cells cannot grow on respiratory carbon sources. To investigate this phenomenon, we studied the effect of cultivation temperature on the efficiency (production of ATP per atom of oxygen consumed, or P/O) of the yeast respiratory chain in glucose-limited chemostats. We determined that even though the specific oxygen consumption rate did not change with temperature, oxygen consumption no longer contributed to mitochondrial ATP generation at temperatures higher than 37 °C. Remarkably, between 30 and 37 °C, we observed a linear increase in respiratory efficiency with growth temperature, up to a P/O of 1.4, close to the theoretical maximum that can be reached in vivo. The temperature-dependent increase in efficiency required the presence of the mitochondrial glycerol-3-phosphate dehydrogenase GUT2. Respiratory chain efficiency was also altered in response to changes in oxygen availibility. Our data show that, even in the absence of alternative oxidases or uncoupling proteins, yeast has retained the ability to dynamically regulate the efficiency of coupling of oxygen consumption to proton translocation in the respiratory chain in response to changes in the environment.
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