- Volume 149, Issue 1, 2003
Volume 149, Issue 1, 2003
- Pathogens And Pathogenicity
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Application of a novel multi-screening signature-tagged mutagenesis assay for identification of Klebsiella pneumoniae genes essential in colonization and infection
More LessKlebsiella pneumoniae is a common cause of urinary tract infections (UTIs) and pneumonia, especially in immunocompromised individuals. Epidemiological studies have revealed that K. pneumoniae infections are frequently preceded by gastrointestinal colonization and the gastrointestinal tract is believed to be the most important reservoir for transmission of the bacteria. To identify genes involved in the ability of K. pneumoniae to colonize the intestine and infect the urinary tract, a novel multi-screening signature-tagged mutagenesis (MS-STM) assay was implemented. In the MS-STM assay, PCR-amplified tags present in the inoculum as well as recovered pools from each infection model are simultaneously subjected to hybridization using each specific tag as a probe. Therefore, screenings of a mutant library in more than one infection model is significantly eased compared to the traditional signature-tagged mutagenesis methodology. From a total of 1440 K. pneumoniae transposon mutants screened, 13 mutants were identified as attenuated in intestinal colonization as well as the UTI model. In addition, six mutants attenuated only in the UTI model were identified. Transposon insertion sites in attenuated mutants were, among others, in genes encoding well-known K. pneumoniae virulence factors such as lipopolysaccharide and capsule, as well as in genes of unknown function.
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A novel ColV plasmid encoding type IV pili
More LessMany septicaemic Escherichia coli strains harbour ColV virulence plasmids. This paper describes pO78V, a conjugative ColV plasmid from an avian pathogenic E. coli strain that encodes type IV pili in addition to other virulence-related genes and tetracycline resistance. Plasmid location of type IV pili genes was demonstrated using Southern hybridization and expression of the pili was demonstrated using RT-PCR and phage sensitivity assays. This is a first report of a ColV plasmid encoding type IV pili. Plasmid pO78V is a mosaic plasmid containing replicons and other genes typical to both IncI1 and IncFII groups. As type IV pili of Gram-negative bacteria are involved in several stages of infection, their presence on a ColV virulence plasmid could expand the repertoire of pathogenesis-related genes.
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Characterization of LppS, an adhesin of Mycoplasma conjunctivae
More LessA serine-rich membrane protein named LppS from Mycoplasma conjunctivae, the aetiological agent of infectious keratoconjunctivitis (IKC) of domestic and wild Caprinae, was characterized. Gene cloning and sequence analysis of the lppS gene revealed that it encoded a membrane protein precursor. The protein had a typical signal sequence and a signal peptidase II cleavage site followed by a cysteine residue representing a potential acylation site. The mature LppS protein had an apparent molecular mass of 150 kDa and was found in the detergent-associated fraction of Tween 20 extracted M. conjunctivae proteins. It possessed a serine-rich domain of 41 aa with 37 (90·2 %) serine residues. Twenty-seven of these serine residues were contiguous. The protein adhered to lamb joint synovial cells. Using an in vitro adhesion model, Fab fragments from IgG directed against recombinant purified LppS were shown to specifically inhibit adhesion of M. conjunctivae to lamb cells. Thus, LppS is likely to be an adhesin of M. conjunctivae that may play an important role in the pathogenesis of IKC.
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Characterization of a chromosomal region of Mycoplasma sp. bovine group 7 strain PG50 encoding a glycerol transport locus (gtsABC)
More LessMycoplasma species bovine group 7, represented by the type strain PG50, is one of six members of the Mycoplasma mycoides cluster and has been implicated in sporadic and outbreak cases of polyarthritis and mastitis in Australian dairy cattle. This study describes cloning and sequencing a 7·9 kb region of the PG50 chromosome and identification of genes involved in glycerol transport (gtsA, gtsB and gtsC) that are followed by a putative lipoprotein gene lppB and a genomic locus containing two ORFs encoding putative membrane proteins. Long range PCR using primers spanning gtsABC and downstream flanking genes, and Southern hybridization analyses using a suite of probes derived from M. mycoides subsp. mycoides small colony type (SC) strain Afadé for gtsA, gtsB and gtsC, lppB and the two downstream genes confirmed that these genes were conserved among Mycoplasma sp. bovine group 7 isolates and mycoplasmas belonging to the M. mycoides subcluster [M. mycoides subsp. mycoides SC, M. mycoides subsp. mycoides large colony type (LC) and M. mycoides subsp. capri] but were absent in mycoplasmas belonging to the Mycoplasma capricolum subcluster (M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae). M. capricolum subsp. capricolum type strain California kid did not hybridize with the probe for gtsA and gave only weak or no hybridization signals with probes derived from the loci downstream of gtsABC, suggesting that this region has diverged in mycoplasmas belonging to subspecies of M. capricolum. It is shown that PG50, after the addition of a physiological concentration of glycerol to the growth medium, generates H2O2 at levels comparable with strain Afadé, implying that the glycerol transport system is functional in Mycoplasma sp. bovine group 7. This suggests that in PG50, as in M. mycoides subsp. mycoides SC, glycerol uptake is followed by phosphorylation to glycerol 3-phosphate and then conversion to dihydroxyacetone phosphate, catalysed by l-α-glycerophosphate oxidase, resulting in the production of H2O2. The ability of Mycoplasma sp. bovine group 7 to generate significant amounts of hydrogen peroxide may be important in pathogenesis.
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Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts
More LessExpressed sequence tag (EST) libraries for Metarhizium anisopliae, the causative agent of green muscardine disease, were developed from the broad host-range pathogen Metarhizium anisopliae sf. anisopliae and the specific grasshopper pathogen, M. anisopliae sf. acridum. Approximately 1700 5′ end sequences from each subspecies were generated from cDNA libraries representing fungi grown under conditions that maximize secretion of cuticle-degrading enzymes. Both subspecies had ESTs for virtually all pathogenicity-related genes cloned to date from M. anisopliae, but many novel genes encoding potential virulence factors were also tagged. Enzymes with potential targets in the insect host included proteases, chitinases, phospholipases, lipases, esterases, phosphatases and enzymes producing toxic secondary metabolites. A diverse array of proteases composed 36 % of all M. anisopliae sf. anisopliae ESTs. Eighty percent of the ESTs that could be clustered into functional groups had significant matches (E<10−5) in other ascomycete fungi. These included genes reported to have specific roles in pathogens with plant or vertebrate hosts. Many of the remaining ESTs had their best blast match among animal, plant and bacterial sequences. These include genes with plant and microbial counterparts that produce potent antimicrobials. The abundance of transcripts discovered for different functional groups varied between the two subspecies of M. anisopliae in a manner consistent with ecological adaptations of the two pathogens. By hastening gene discovery this project has enhanced development of improved mycoinsecticides. In addition, the M. anisopliae ESTs represent a significant contribution to the extensive database of sequences from ascomycetes that are saprophytes or plant and vertebrate pathogens. Comparative analyses of these sequences is providing important information about the biology and evolutionary history of this clade.
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Differential expression of Candida albicans phospholipase B (PLB1) under various environmental and physiological conditions
More LessCandida is the fourth most common organism responsible for bloodstream infections in many intensive care units, with Candida albicans being the most predominant species isolated in such cases. It has previously been shown that candidal phospholipase B, encoded by the PLB1 gene, is an important virulence factor for C. albicans pathogenesis. In this study, the effects of environmental factors (carbohydrate source and pH) and physiological conditions (serum, phospholipids and temperature) on the expression of PLB1 by C. albicans cells grown in rich [Sabouraud dextrose broth (SB) or yeast extract/peptone/dextrose] or chemically defined [Lee's, RPMI-1640 or yeast nitrogen base (YNB)] media were investigated. Northern blot analyses revealed that PLB1 mRNA was expressed in C. albicans cells grown in rich media at 30 °C but not at 37 °C. However, the protein Plb1p was detected in fungal cells growing at 37 °C in SB, as determined by Western blot analysis, indicating that although the mRNA for this gene was not detected, the actual gene product was present at this temperature. Expression of PLB1 was detected in cells grown in YNB/glucose at 30 °C but not at 37 °C. However, growth of C. albicans in YNB/glucose supplemented with serum and phospholipids resulted in expression of PLB1 at 37 °C also. Additionally, acidic pH induced higher levels of PLB1 mRNA expression compared to neutral pH, while the morphological form of C. albicans did not have any influence on the expression of this gene. The studies described here show that the expression of PLB1 is regulated by nutritional supplementation, environmental factors and the growth phase of the C. albicans cells, as well as by physiological conditions. The differential expression of PLB1 in response to environmental factors may be correlated to host-specific components available to C. albicans during infection.
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- Plant-Microbe Interactions
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Surface motility in Pseudomonas sp. DSS73 is required for efficient biological containment of the root-pathogenic microfungi Rhizoctonia solani and Pythium ultimum
Pseudomonas sp. DSS73 was isolated from the rhizoplane of sugar beet seedlings. This strain exhibits antagonism towards the root-pathogenic microfungi Pythium ultimum and Rhizoctonia solani. Production of the cyclic lipopeptide amphisin in combination with expression of flagella enables the growing bacterial culture to move readily over the surface of laboratory media. Amphisin is a new member of a group of dual-functioning compounds such as tensin, viscosin and viscosinamid that display both biosurfactant and antifungal properties. The ability of DSS73 to efficiently contain root-pathogenic microfungi is shown to arise from amphisin-dependent surface translocation and growth by which the bacterium can lay siege to the fungi. The synergistic effects of surface motility and synthesis of a battery of antifungal compounds efficiently contain and terminate growth of the microfungi.
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Volumes and issues
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