Dayle A. Daines

VapC-1 of Nontypeable Haemophilus influenzae Is a Ribonuclease

Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.

Evidence for Multimerization of Neu Proteins Involved in Polysialic Acid Synthesis in Escherichia coli K1 Using Improved LexA-Based Vectors

Recently, M. Dmitrova et al. (Mol. Gen. Genet. 257:205-212, 1998) described a LexA-based genetic system to monitor protein-protein interactions in an Escherichia coli background. However, the plasmids used in this system, pMS604 and pDP804, were not readily amenable for general use. In this report, we describe modifications of both plasmids that allow fragments of DNA to be fused to either vector in any reading frame. Homodimerization and heterodimerization of full-length proteins involved in polysialic acid synthesis in E. coli K1, as well as heterodimerization between a full-length protein and a protein fragment, demonstrate the usefulness of the modified plasmids for investigating bacterial protein-protein interactions in vivo.

Chemically Defined Media for Growth of Haemophilus influenzae Strains

ABSTRACT A chemically defined medium that supports the growth of both encapsulated and nontypeable Haemophilus influenzae strains in broth to densities that are ≥ 109 CFU/ml or on agar plates is described. The mean generation time of a panel of clinical isolates was comparable to that in rich, chemically undefined media (brain-heart infusion broth supplemented with heme and β-NAD).

The NeuC Protein of Escherichia coli K1 Is a UDP N-Acetylglucosamine 2-Epimerase

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an alpha-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (DeltaneuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements DeltaneuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of [2-(14)C]acetamidoglucal and [N-(14)C]acetylmannosamine (ManNAc) from UDP-[(14)C]GlcNAc. The formation of reaction intermediate 2-acetamidoglucal with the concomitant release of UDP was confirmed by proton and phosphorus nuclear magnetic resonance spectroscopy. NeuC could not use GlcNAc as a substrate. These data suggest that neuC encodes an epimerase that catalyzes the formation of ManNAc from UDP-GlcNAc via a 2-acetamidoglucal intermediate. The unexpected release of the glucal intermediate and the extremely low rate of ManNAc formation likely were a result of the in vitro assay conditions, in which a key regulatory molecule or protein was absent.

Identification and characterization of a nontypeable Haemophilus influenzae putative toxin-antitoxin locus

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.

Use of LexA-based system to identify protein-protein interactions in vivo

Publisher Summary This chapter describes a system for analyzing both homo- and heterodimerization of full-length proteins and protein fragments in an Escherichia coli background. The characterization of protein-protein interactions can reveal much about the structure and function of individual proteins and protein complexes. Many methods designed to identify these interactions have been reported, including yeast two hybrid and plasmid-based bacterial systems. This system has been successful in determining interactions between proteins involved in the biosynthesis of the polysialic acid capsule of E. coli K1. These interactions include those between full-length proteins as well as those between full-length proteins and protein fragments. However, it is not necessary to restrict the systems use to studying only Escherichia coli protein interactions. Another feature of this system is that the detection of interaction between fusions is not disrupted by the tandem expression of unfused subunits in the host strain. Chloramphenicol acetyltransferase (CAT) fused to the wild-type LexA DNA binding domain(DBD) efficiently repressed lacZ transcription in SU101, even though this strain carries a transposon that expresses subunits of the same type I CAT. This data also illustrates that some multimeric proteins can be studied using this system, since CAT is a well-characterized homotrimer.

H. influenzae Consortium: integrative study of H. influenzae-human interactions.

Developments in high-throughput analysis tools coupled with integrative computational techniques have enabled biological studies to reach new levels. The ability to correlate large volumes of diverse data types into cohesive models of organism function has spawned a new systematic approach to biological investigation. The creation of a new consortium has been proposed to investigate a single organism utilizing these comprehensive approaches. The Haemophilus influenzae Consortium (HIC) would be comprised of five laboratories, each providing separate and complementary areas of expertise in the study of Haemophilus influenzae (HI). The 5-year study proposes to develop coherent models of HI, both as a stand-alone organism, and more importantly, as a human pathogen. Studies in growth condition specificity followed by genomic, metabolic, and proteomic experimentation will be combined and integrated through computational and experimental analyses to form dynamic and predictive models of HI and its responses. Data from the HIC will allow greater understanding of cellular behavior, pathogen-host interactions, bacterial infection, and provide future scientific endeavors with a template for studies of other pathogens.