Identification and characterization of a nontypeable Haemophilus influenzae putative toxin-antitoxin locusReportar como inadecuado

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BMC Microbiology

, 4:30

First Online: 26 July 2004Received: 15 May 2004Accepted: 26 July 2004


BackgroundCertain strains of an obligate parasite of the human upper respiratory tract, nontypeable Haemophilus influenzae NTHi, can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NTHi survival inside human cells, v irulence-a ssociated protein D vapDHi, encoded by gene HI0450. Both vapDHiand a flanking gene, HI0451, exhibit the genetic and physical characteristics of a toxin-antitoxin TA locus, with VapDHiserving as the toxin moiety and HI0451 as the antitoxin. We propose the name VapXHifor the HI0451 antitoxin protein. Originally identified on plasmids, TA loci have been found on the chromosomes of a number of bacterial pathogens, and have been implicated in the control of translation during stressful conditions. Translation arrest would enhance survival within human cells and facilitate persistent or chronic mucosal infections.

ResultsIsogenic mutants in vapDHiwere attenuated for survival inside human respiratory epithelial cells NCI-H292 and human brain microvascular endothelial cells HBMEC, the in vitro models of mucosal infection and the blood-brain barrier, respectively. Transcomplementation with a vapDHiallele restored wild-type NTHi survival within both cell lines. A PCR survey of 59 H. influenzae strains isolated from various anatomical sites determined the presence of a vapDHiallele in 100% of strains. Two isoforms of the gene were identified in this population; one that was 91 residues in length, and another that was truncated to 45 amino acids due to an in-frame deletion. The truncated allele failed to transcomplement the NTHi vapDHisurvival defect in HBMEC. Subunits of full-length VapDHihomodimerized, but subunits of the truncated protein did not. However, truncated protein subunits did interact with full-length subunits, and this interaction resulted in a dominant-negative phenotype. Although Escherichia coli does not contain a homologue of either vapDHior vapXHi, overexpression of the VapDHitoxin in trans resulted in E. coli cell growth arrest. This arrest could be rescued by providing the VapXHiantitoxin on a compatible plasmid.

ConclusionWe conclude that vapDHiand vapXHimay constitute a H. influenzae TA locus that functions to enhance NTHi survival within human epithelial and endothelial cells.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2180-4-30 contains supplementary material, which is available to authorized users.

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Autor: Dayle A Daines - Justin Jarisch - Arnold L Smith


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