Parameters Associated with Cloning in Actinobacillus actinomycetemcomitans

Galli, Dominique M.; Kerr, Micah S.; Fair, Amber D.; Permpanich, Piyanuj; LeBlanc, Donald J.

doi:10.1006/plas.2001.1556

Cited by 6 publications

(6 citation statements)

References 28 publications

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“…Subsequently, a major portion of the ltx A gene was deleted as a 1 kb fragment by NcoI digest and religation. Next a 1.3 kb kanamycin resistance cassette retrieved from pMK3 (Galli et al ., 2001b) was inserted into this NcoI site. The final construct was then introduced into VT726S by electroporation.…”

Section: Methodsmentioning

confidence: 99%

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils

2006

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SummaryNeutrophils are initially the predominant cells involved in the host defence of bacterial infections, including periodontal disease. Aggressive periodontitis is associated with Actinobacillus actinomycetemcomitans , a Gram-negative capnophilic microorganism. Infections caused by A. actinomycetemcomitans are not resolved by the host immune response despite the accumulation of neutrophils at the site of inflammation. To better understand the role of natural host defence mechanisms in A. actinomycetemcomitans infections, the interaction of phenotypically diverse strains of this pathogen with human neutrophils was assessed directly using techniques such as genetic labelling with the gene for green fluorescent protein, fluorescence-activated cell sorting and fluorescence imaging. The study included clinical isolates of A. actinomycetemcomitans represented by self-aggregating, biofilm-associated and isogenic planktonic variants. Data obtained showed that complement-mediated phagocytosis of A. actinomycetemcomitans was generally inefficient regardless of strain-specific serotype or leukotoxin production. Furthermore, the majority of ingested bacteria remained viable after exposure to neutrophils for 1 h. Interestingly, uptake of antibodyopsonized bacteria resulted in the rapid cell death of neutrophils. This was in contrast to ingestion of complement-opsonized bacteria, which did not affect neutrophil viability. The methods used in this study provided reliable and reproducible results with respect to adherence, phagocytosis and killing of A. actinomycetemcomitans when encountering human neutrophils.

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Section: Methodsmentioning

confidence: 99%

Resistance of fluorescent-labelled Actinobacillus actinomycetemcomitans strains to phagocytosis and killing by human neutrophils

2006

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“…For strains HK912, HK988 and HK1002 we did not obtain mutants with the resistancemarker gene integrated into the chromosomal hgpA gene, whereas for strain HK989 several such mutants were recovered. Differences in transformability among A. actinomycetemcomitans strains have been reported previously (Galli et al, 2002). Notably, in contrast to the parental strain HK989, the mutated version of strain HK989 was unable to grow on human haemoglobin The experiments were performed as described in Fig.…”

Section: Insertional Inactivation Of the Hgpa Homologuementioning

confidence: 96%

Differences in iron acquisition from human haemoglobin among strains of Actinobacillus actinomycetemcomitans The GenBank accession numbers for the tpbA homologue sequences reported in this paper are AY028441 (strain HK1119), AF359437 (HK912), AF359438 (HK989), AF359439 (HK1002), AF359440 (HK988) and AF359441 (HK961); the GenBank accession numbers for the hgpA homologue sequences reported in this paper are AF359442 (HK989), AF359443 (HK988), AF359444 (HK981), AF359445 (HK961), AF359446 (JP2), AF359447 (HK91

2002

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To get a better insight into the physiology of the high-toxic JP2 clone of Actinobacillus actinomycetemcomitans serotype b, which is strongly associated with juvenile periodontitis in adolescents of African descent, the modes of iron acquisition in this clone were examined and compared to those of other strains of the species. None of the strains examined could utilize human transferrin as a source of iron. This was in accordance with the presence of a non-functional tbpA gene, which normally encodes the A subunit of the transferrin-bindingprotein complex. Southern blot analysis indicated that functional duplications of tbpA were not present in the genome. Thus, A. actinomycetemcomitans seems to be in a process of evolution, in which iron acquisition from host transferrin is not essential as in many other members of the Pasteurellaceae. All strains could utilize haem as a source of iron. All 11 A. actinomycetemcomitans strains examined harboured a single genomic sequence with homology to the hgpA gene encoding haemoglobin-binding protein A in Haemophilus influenzae. However, in all three strains belonging to the JP2 clone and in one serotype e strain hgpA was a pseudogene. Seven other strains possessed a functional hgpA gene which, according to insertion mutagenesis experiments, was responsible for the ability of these strains to utilize haemoglobin as a source of iron. Thus, the presence of an hgpA pseudogene and the inability to use human haemoglobin as an iron source discriminate the high-toxic JP2 clone from low-toxic serotype b strains and most other strains of A. actinomycetemcomitans.

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“…The development of an electro‐transformation system and the construction of shuttle plasmids for A. actinomycetemcomitans (37) have enhanced the molecular analysis of virulence factors (4, 11, 18, 23, 24, 36, 37). Unfortunately, inefficient transformation is frequently encountered (9, 21, 37). Several factors that influence the efficiency of transformation, such as host modification, plasmid DNA concentration, bacterial cell density, pH, length of recovery time, freezing, and plasmid size have been identified (9, 37).…”

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“…Unfortunately, inefficient transformation is frequently encountered (9, 21, 37). Several factors that influence the efficiency of transformation, such as host modification, plasmid DNA concentration, bacterial cell density, pH, length of recovery time, freezing, and plasmid size have been identified (9, 37). Moreover, it has been observed that phenotypic variation enhances the electro‐transformation efficiency of the strains, suggesting that rough isolates are more difficult to transform than smooth variants (9).…”

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confidence: 99%

“…Several factors that influence the efficiency of transformation, such as host modification, plasmid DNA concentration, bacterial cell density, pH, length of recovery time, freezing, and plasmid size have been identified (9, 37). Moreover, it has been observed that phenotypic variation enhances the electro‐transformation efficiency of the strains, suggesting that rough isolates are more difficult to transform than smooth variants (9). As a consequence, rough A. actinomycetemcomitans have been refractory to genetic analysis (39) and virulence factors have been almost exclusively investigated in smooth strains.…”

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confidence: 99%

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