Mutations in HlyD, Part of the Type 1 Translocator for Hemolysin Secretion, Affect the Folding of the Secreted Toxin

Pimenta, Aparecida Linhares; Racher, Kathleen I.; Jamieson, Lee; Blight, Mark A.; Holland, I. B.

doi:10.1128/jb.187.21.7471-7480.2005

Cited by 65 publications

(66 citation statements)

References 53 publications

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“…One island encodes an ABC transporter and a probable hemolysin translocator. In E. coli, hemolysin translocator has been demonstrated to be essential for the secretion of the Repeats-in-Toxin (RTX) hemolytic toxin HlyA (Pimenta et al, 2005). Several putative Repeats-in-Toxin proteins have been identified in the V. nigripulchritudo genomes but were not restricted to HP strains (not shown).…”

Section: Discussionmentioning

confidence: 99%

Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits

et al. 2013

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Vibrio nigripulchritudo is an emerging pathogen of farmed shrimp in New Caledonia and other regions in the Indo-Pacific. The molecular determinants of V. nigripulchritudo pathogenicity are unknown; however, molecular epidemiological studies have suggested that pathogenicity is linked to particular lineages. Here, we performed high-throughput sequencing-based comparative genome analysis of 16 V. nigripulchritudo strains to explore the genomic diversity and evolutionary history of pathogen-containing lineages and to identify pathogen-specific genetic elements. Our phylogenetic analysis revealed three pathogen-containing V. nigripulchritudo clades, including two clades previously identified from New Caledonia and one novel clade comprising putatively pathogenic isolates from septicemic shrimp in Madagascar. The similar genetic distance between the three clades indicates that they have diverged from an ancestral population roughly at the same time and recombination analysis indicates that these genomes have, in the past, shared a common gene pool and exchanged genes. As each contemporary lineage is comprised of nearly identical strains, comparative genomics allowed differentiation of genetic elements specific to shrimp pathogenesis of varying severity. Notably, only a large plasmid present in all highly pathogenic (HP) strains encodes a toxin. Although less/non-pathogenic strains contain related plasmids, these are differentiated by a putative toxin locus. Expression of this gene by a non-pathogenic V. nigripulchritudo strain resulted in production of toxic culture supernatant, normally an exclusive feature of HP strains. Thus, this protein, here termed 'nigritoxin', is implicated to an extent that remains to be precisely determined in the toxicity of V. nigripulchritudo.

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Section: Discussionmentioning

confidence: 99%

Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits

et al. 2013

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“…B. Holland, unpublished data). On the other hand, significant amounts of secreted HlyA remain associated with the cell envelope, accessible to extracellular trypsin, in strains expressing hlyBD (Pimenta et al, 2005). When wild-type HlyA and HlyA99 were compared in the presence of HlyBD, high levels of the wild-type protein were found to be cell-associated, with apparently even higher levels detected in the case of the mutant HlyA99 protein (Fig.…”

Section: Localization Of Cell-associated Wild-type Hlya and Hlya99 Prmentioning

confidence: 97%

“…These were carried out as described previously (Pimenta et al, 2005). In the titration method, one haemolytic unit was defined as the amount of HlyA necessary to produce 50 % lysis of the erythrocytes.…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, we have shown that HlyA molecules with reduced activity due to mutations in HlyD can be restored to full activity following denaturation and then renaturation (Pimenta et al, 2005). Wild-type haemolysin, HlyA99 and HlyA Del6 in culture supernatants were therefore concentrated by precipitation by ammonium sulphate, denatured by resuspending in 6 M GnCl and renatured by dilution into assay buffer (see Methods) to measure haemolytic activity.…”

Section: Kdahlya Proteinmentioning

confidence: 99%

“…HlyA is then transported directly to the cell surface through the HlyB : HlyD : TolC trans-envelope tunnel, where the restricted diameter of TolC (Andersen et al, 2002) indicates that HlyA must be translocated as an unfolded polypeptide chain. Previous evidence suggests that HlyA molecules can then proceed to the external medium by two distinct mechanisms: directly to the medium as the free protein or following accumulation in a trypsin-accessible form on the cell surface (Pimenta et al, 2005) with a final release step in association with outer membrane vesicles (Balsalobre et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

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Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin

et al. 2010

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Escherichia coli haemolysin A (HlyA), an RTX toxin, is secreted probably as an unfolded intermediate, by the type I (ABC transporter-dependent) pathway, utilizing a C-terminal secretion signal. However, the mechanism of translocation and post-translocation folding is not understood. We identified a mutation (hlyA99) at the extreme C terminus, which is dominant in competition experiments, blocking secretion of the wild-type toxin co-expressed in the same cell. This suggests that unlike recessive mutations which affect recognition of the translocation machinery, the hlyA99 mutation interferes with some later step in secretion. Indeed, the mutation reduced haemolytic activity of the toxin and the activity of b-lactamase when the latter was fused to a Cterminal 23 kDa fragment of HlyA carrying the hlyA99 mutation. A second mutant (hlyAdel6), lacking the six C-terminal residues of HlyA, also showed reduced haemolytic activity and neither mutant protein regained normal haemolytic activity in in vitro unfolding/refolding experiments. Tryptophan fluorescence spectroscopy indicated differences in structure between the secreted forms of wild-type HlyA and the HlyA Del6 mutant. These results suggested that the mutations affected the correct folding of both HlyA and the b-lactamase fusion. Thus, we propose a dual function for the HlyA C terminus involving an important role in post-translocation folding as well as targeting HlyA for secretion.

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Bacterial Multidrug Transporters: Molecular and Clinical Aspects

Lomovskaya

Zgurskaya

Bostian

2009

Transporters as Drug Carriers

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Mutations in HlyD, Part of the Type 1 Translocator for Hemolysin Secretion, Affect the Folding of the Secreted Toxin

Cited by 65 publications

References 53 publications

Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits

Comparative genomics of pathogenic lineages of Vibrio nigripulchritudo identifies virulence-associated traits

Mutations affecting the extreme C terminus of Escherichia coli haemolysin A reduce haemolytic activity by altering the folding of the toxin

Bacterial Multidrug Transporters: Molecular and Clinical Aspects

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