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Promoterless recombinant adeno-associated virus is used without nucleases to target the human coagulation factor IX gene to the liver-expressed albumin locus in haemophilia B mice, with an on-target integration into ~0.5% of the albumin alleles in hepatocytes; stable F9 plasma levels at 7-20% of normal were obtained, leading to normal coagulation times in treated factor-IX-deficient mice. Nuclease-free genome targeting less risky Most genome editing applications under consideration for therapeutic use require the presence of a site-specific endonuclease, but the uncontrolled presence of endonucleases in tissues can potentially cause significant adverse effects. Here, Mark Kay and colleagues describe the use of promoterless recombinant adeno-associated virus to target -- without the need for nucleases -- the human coagulation factor IX gene to the liver-expressed albumin locus in haemophilia B mice. The approach achieves on-target integration into about 0.5% of the albumin alleles in hepatocytes. Stable F9 plasma levels in treated factor IX deficient mice were 7-20% of normal, leading to coagulation times within the normal range. Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion.sup.1 and oncogenesis.sup.2. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy.sup.3. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target.sup.4 and off-target.sup.5,6; and oncogene activation caused by promoter integration, even without nucleases.sup.7. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into ~0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration., Author(s): A. Barzel [sup.1] , N. K. Paulk [sup.1] , Y. Shi [sup.2] , Y. Huang [sup.1] , K. Chu [sup.1] , F. Zhang [sup.1] , P. N. Valdmanis [sup.1] [...]

The Haemophilus influenzae ORF designated HI1275 in the Rd KW20 genomic sequence encodes a putative S-adenosyl methyltransferase with significant similarity to tellurite-resistance determinants (tehB) in other species. While the H. influenzae tehB can complement an Escherichia coil tehB mutation, thus restoring tellurite resistance, its role in H. influenzae is unknown. In a previous study defining the iron and haem modulon of H. influenzae, we showed that transcription of this gene in H. influenzae Rd KW20 increases during growth in iron- and haem-restricted media. Since iron and haem uptake genes, and other known virulence factors, constitute the majority of the iron- and haem-regulated gene set, we postulated that tehB may play a role in nutrient acquisition and/or the virulence of H. influenzae. A tehB mutant was constructed in the H. influenzae type b strain 10810 and was evaluated for growth defects in various supplemented media, as well as for its ability to cause infection in rat models of infection. Deletion of tehB leads to an increase in sensitivity both to tellurite and to the oxidizing agents cumene hydroperoxide, tert-butyl hydroperoxide and hydrogen peroxide. The tehB mutant additionally showed a significantly reduced ability to utilize free haem as well as several haem-containing moieties including haem--human serum albumin, haemoglobin and haemoglobin--haptoglobin. Examination of the regulation kinetics indicated that transcription of tehB was independent of both tellurite exposure and oxidative stress. Paired comparisons of the tehB mutant and the wild-type H. influenzae strain 10810 showed that tehB is required for wild-type levels of infection in rat models of H. influenzae invasive disease. To our knowledge this is the first report of a role for tehB in virulence in any bacterial species. These data demonstrate that H. influenzae tehB plays a role in both resistance to oxidative damage and haem uptake/utilization, protects H. influenzae from tellurite exposure, and is important for virulence of this organism in an animal model of invasive disease. DOI 10.1099/mic.0.036400-0

Actinobacillus actinomycetemcomitans is naturally competent for transformation, with a transformation system similar to that of Haemophilus influenzae that preferentially takes up DNA bearing uptake signal sequences (USS) with the same 9-base USS core. This study examined the function of the extended 29-base USS, which comprises a highly conserved 1st region (containing the 9-base core) and 2nd and 3rd semi-conserved AT-rich regions, in transformation of A. actinomycetemcomitans. Transformation frequency was not affected by either location (in middle or at 5' end) or quantity (one or two) of USS in donor DNA. Relative transformation efficiencies (in comparison to the positive control) were 28-67% for linear DNA with single-base mutations in the USS 1st region, and 47% and 73%, respectively, for linear DNA with USS that contained either a non-consensus 2nd or a non-consensus 3rd region. Plasmids with a stand-alone 1st or a stand-alone 2nd-3rd region exhibited 21% and 6% relative transformation efficiencies, respectively. It was also noted that A. actinomyceterncomitans and H. influenzae were similar in the frequencies and distribution patterns of USS in their genomes. In conclusion, all three regions of the extended 29-base USS are required for optimum transformation in A. actinomyceterncomitans.

Haemophilus influenzae is an important cause of human disease, and its lipopolysaccharide (LPS) is known to be a major virulence factor. H. influenzae produces short-chain LPS of which the heterogeneity is often visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using silver staining for detection. Individual bands have not previously been recovered by this method in quantities sufficient for mass spectrometry. In an attempt toward the development of sensitive mass spectrometrical strategies to be used in structural studies of H. influenzae LPS and LPS from other bacteria, we have applied here our previously described slab-PAGE-based micropurification method to obtain unmodified LPS fractions of high purity (>95%) from a crude LPS preparation of H.influenzae strain Rd. Two LPS-fractions were obtained which, after a procedure including mild acid hydrolysis, dephosphorylation, and permethylation of the resulting oligosaccharides, were subjected to tandem electrospray ionization mass spectrometry (ESI-MS/MS). The quantities of micropurified LPS fractions--the recovery of LPS in terms of total mass was 30%--were found sufficient to allow the characterization of LPS glycoforms. The ESI-MS spectra of the individual bands showed reduced heterogeneity. Furthermore, the integrity of the micropurified LPS was confirmed. The spectra-displayed molecular ions showed improved intensity, increased respective signal-to-noise ratios demonstrating the sensitivity of analysis. Consequently, both the direct determination of the molecular masses of the gel-separated LPS glycoforms and sequence analyses using ESI-MS/MS were possible.

Byline: J.A. Garcia-Rodriguez (1), F. Baquero (2), J. Garcia de Lomas (3), L. Aguilar (4) Abstract: A Haemophilus influenzae multicenter antimicrobial susceptibility surveillance was carried out. An overall [beta],-lactamase production rate of 25.7% was found, with significant differences between hospitals ranging between 7.9 and 47.9%. Presumable intrinsic resistance was found in 4.6% of strains. No differences in susceptibility were observed between [beta]-lactamase positive and [beta]-lactamase negative strains for all antibiotics, except for penicillin, amoxicillin and cefaclor. A higher prevalence of resistance was found for clarithromycin vs azithromycin (22.6 vs 0.7%). Differences in all these factors make surveillance essential as a guideline for empiric treatment in community-acquired respiratory tract infections. Author Affiliation: (1) University Hospital, Po de San Vicente 108, E-37007 Salamanca, Spain, ES (2) Ramon y Cajal Hospital, Crtra. De Colmenar Km.1, E-28034 Madrid, Spain, ES (3) Instituto Valenciano de Microbiologia, Crtra. De Berera a S. Antonio, Km. 0.3, E-46117 Betera, Valencia, Spain, ES (4) Departamento Medico. Smithkline Beecham Pharmaceuticals, Valle de la Fuenfria 3, E-28034 Madrid, Spain, ES Article note: Received: November 17, 1998 * Revision accepted: April 4, 1999

We studied genetic diversity in Streptococcus pneumoniae and Haemophilus influenzae in throat culture isolates from 38 children attending two day-care centers in Michigan. Culture specimens were collected weekly; 184 S. [...]

Haemophilus influenzae type b (Hib) infections in California children fell dramatically from 1990 to 1996, after an Hib vaccine was introduced. Hib infections can cause meningitis, pneumonia, and infectious arthritis. Invasive Hib disease in children under 5 years dropped 95% nationwide from 1989 to 1995, and dropped 99% in California from 1990 to 1996. The type b viral serotype caused 58% of infections in California. Continued surveillance of both infection rates and the viral types causing infection will allow more accurate evaluation of vaccination programs.