Identification and characterization of a nontypeable Haemophilus influenzae putative toxin-antitoxin locus -: art. no. 30

Background: Certain 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, virulence-associated protein D (vapD(Hi), encoded by gene H10450). Both vapD(Hi) and a flanking gene, H10451, exhibit the genetic and physical characteristics of a toxin/antitoxin ( TA) locus, with VapD(Hi) serving as the toxin moiety and H10451 as the antitoxin. We propose the name VapX(Hi) for the H10451 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. Results: Isogenic mutants in vapD(Hi) were 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 vapD(Hi) allele 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 vapD(Hi)allele 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 vapD(Hi) survival defect in HBMEC. Subunits of full-length VapD(Hi) homodimerized, 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 vapD(Hi) or vapX(Hi), overexpression of the VapD(Hi) toxin in trans resulted in E. coli cell growth arrest. This arrest could be rescued by providing the VapX(Hi) antitoxin on a compatible plasmid. Conclusion: We conclude that vapDHi and vapX(Hi) may constitute a H. influenzae TA locus that functions to enhance NTHi survival within human epithelial and endothelial cells.