BackgroundNontypeable Haemophilus influenzae (NTHi) is a significant human pathogen responsible for respiratory tract infections and the most common cause of recurrent otitis media. Type II toxin-antitoxin (TA) systems are genetic elements that code for a stable protein toxin and a labile antitoxin that are thought to be involved in metabolic regulation of bacteria by enabling a switch to a dormant state under stress conditions. The contribution to infection persistence of the NTHi TA loci vapBC-1 and vapXD was examined in this study.ResultsDeletions in vapBC-1, vapXD and vapBC-1 vapXD significantly decreased the survival of NTHi co-cultured with primary human respiratory tissue at the air-liquid interface and in the chinchilla model of otitis media. The TA deletions did not affect the growth dynamics of the mutants in rich media, their ultra-structural morphology, or display appreciable synergy during NTHi infections. The toxin and antitoxin proteins of both pairs heterodimerized in vivo. Consistent with our previous findings regarding the VapC-1 toxin, the NTHi VapD toxin also displayed ribonuclease activity.ConclusionsWe conclude that the vapBC-1 and vapXD TA loci enhance NTHi survival and virulence during infection in vitro and in vivo using a mechanism of mRNA cleavage, and that these conserved TA pairs represent new targets for the prophylaxis and therapy of otitis media and other NTHi-caused mucosal diseases.

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.

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.

A chemically defined medium that supports the growth of both encapsulated and nontypeable Haemophilus influenzae strains in broth to densities that are > 10 9 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 ␤-NAD).Haemophilus influenzae is a fastidious, gram-negative coccus that inhabits the upper respiratory system of humans and has an obligate requirement for heme and ␤-NAD for aerobic growth. Prior investigators studying Haemophilus influenzae sought a chemically defined medium to facilitate genetic and metabolic studies. Multiple defined media were devised for use with Rd derivatives of H. influenzae (2,3,4,5,6,8,13,15), but these media would not support the growth of many nontypeable clinical isolates or encapsulated strains.During the course of experiments studying the invasion of human cell lines by pathogenic H. influenzae, we found that RPMI medium-based tissue culture media appeared to support bacterial growth. Further studies led to the development of the chemically defined media described herein. Table 1 describes the strains used in this study, which were stored at Ϫ70°C in 10% skim milk and were subcultured onto chocolate agar. One liter of chocolate agar was prepared by adding 36 g of GC base (catalog no. 228920; Difco, Detroit, Mich.) and 10 g of hemoglobin (catalog no. ZIZ392; BD Biosciences, Sparks, Md.) and autoclaving at 121°C at 15 lb/in 2 for 20 min; after cooling to 55°C, 5,000 U of bacitracin and 10 ml of GCHI Rehydrating Solution (catalog no. 450411; Remel, Lenexa, Kans.) were added and the plates were poured. Supplemented brain-heart infusion (sBHI) agar was prepared by adding 37 g of BHI media (catalog no. 211059; BD Biosciences) and 15 g of BactoAgar (catalog no. 214530; BD Biosciences) to 1 liter of deionized water, autoclaving, cooling to 55°C, and adding 10 ml of ␤-NAD and 10 ml of heme and L-histidine stock. The heme-histidine stock was prepared by dissolving 0.2 g of L-histidine (freebase; Sigma catalog no. H 8000) in 200 ml of deionized water and then adding 0.2 g of hemin HCl (catalog no. H 5533, bovine; Sigma) and 4 ml of 1 N NaOH and steaming over a boiling water bath for 5 to 10 min to solubilize the mixture. The solution was then cooled to room temperature, filter sterilized (0.2-m pore size), and placed in a foil-covered bottle at 4°C. We found that the histidine decreased the rate at which the hemin precipitated from solution. ␤-NAD ϩ stock was prepared by dissolving 100 mg of ␤-NAD ϩ (catalog no. N 7004; Sigma) in 100 ml of deionized water, filter sterilizing (0.2-m pore size), and storing at 4°C. sBHI broth was prepared without the agar.Defined liquid medium was made with the following: 191 ml of RPMI 1640 with L-glutamine and 25 mM HEPES, pH 7.26 (catalog no. 61870036; InVitrogen), 2 ml of a 100 mM MEM sodium pyruvate solution (catalog no. 11360070; InVitrogen), 2 ml of ␤-NAD ϩ stock, 4 ml of heme-L-histidine ...

Microvascular leakage has been implicated in the pathogenesis of multiple organ dysfunction during trauma. Previous studies suggest the involvement of myosin light chain (MLC) phosphorylation-triggered endothelial contraction in the development of microvascular hyperpermeability. Myosin light chain kinase (MLCK) plays a key role in the control of MLC-phosphorylation status; thus, it is thought to modulate barrier function through its regulation of intracellular contractile machinery. The aim of this study was to further investigate the endothelial mechanism of MLC-dependent barrier injury in burns, focusing on the long isoform of MLCK (MLCK-210) that has recently been identified as the predominant isoform expressed in vascular endothelial cells. An MLCK-210 knockout mouse model was subjected to third-degree scald burn covering 25% total body surface area. The mesenteric microcirculation was observed using intravital microscopy, and the microvascular permeability was assessed by measuring the transvenular flux of fluorescein isothiocyanate-albumin. In a separate experiment, in vivo mesenteric hydraulic conductivity (Lp) was measured using the modified Landis technique. The injury caused a profound microvascular leakage, as indicated by a 2-fold increase in albumin flux and 4-fold increase in Lp at the early stages, which was associated with a high mortality within the 24-h period. Compared with wild-type control, the MLCK-210-deficient mice displayed a significantly improved survival with a greatly attenuated microvascular hyperpermeability response to albumin and fluid. These results provide direct evidence for a role of MLCK-210 in mediating burn-induced microvascular barrier injury and validate MLCK-210 as a potential therapeutic target in the treatment of burn edema.

The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an ␣-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 (⌬neuC), 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 ⌬neuC. 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

Sea level rise and the anthropogenic warming of the world's oceans is not only an environmental tragedy, but these changes also result in a significant threat to public health. Along with coastal flooding and the encroachment of saltwater farther inland comes an increased risk of human interaction with pathogenic Vibrio species, such as Vibrio cholerae, V. vulnificus and V. parahaemolyticus. This minireview examines the current literature for updates on the climatic changes and practices that impact the location and duration of the presence of Vibrio spp., as well as the infection routes, trends and virulence factors of these highly successful pathogens. Finally, an overview of current treatments and methods for the mitigation of both oral and cutaneous exposures are presented.

No abstract