Sensitivity of Haemophilus influenzae to Antibiotics

The use of many different antibiotics to treat chest infection has led us to test the sensitivity of 68 strains of Haemophilus influenzae to 15 different compounds. These included established compounds such as ampicillin and tetracycline and newer agents such as cephalosporins and clindamycin. The minimum inhibitory concentrations of the compounds for H. influenzae were then compared with blood levels attained after the usual dose regimens. There has been a significant increase in tetracycline resistance in the last few years, but all strains were sensitive to ampicillin, chloramphenicol, sulphamethoxazole, and trimethoprim, Several antibiotics were found to be microbiologically unsuitable for treating H. influenzae infections.     

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.     

Insights into substrate gating in H. influenzae rhomboid

Rhomboids are a remarkable class of serine proteases that are embedded in lipid membranes. These membrane-bound enzymes play key roles in cellular signaling events, and disruptions in these events can result in numerous disease pathologies, including hereditary blindness, type 2 diabetes, Parkinson's disease, and epithelial cancers. Recent crystal structures of rhomboids from Escherichia coli have focused on how membrane-bound substrates gain access to a buried active site. In E. coli, it has been shown that movements of loop 5, with smaller movements in helix 5 and loop 4, act as substrate gate, facilitating inhibitor access to rhomboid catalytic residues. Herein we present a new structure of the Haemophilus influenzae rhomboid hiGlpG, which reveals disorder in loop 5, helix 5, and loop 4, indicating that, together, they represent mobile elements of the substrate gate. Substrate cleavage assays by hiGlpG with amino acid substitutions in these mobile regions demonstrate that the flexibilities of both loop 5 and helix 5 are important for access of the substrates to the catalytic residues. Mutagenesis indicates that less mobility by loop 4 is required for substrate cleavage. A reexamination of the reaction mechanism of rhomboid substrates, whereby cleavage of the scissile bond occurs on the si-face of the peptide bond, is discussed.     

Host specificity of DNA in Haemophilus influenzae: DNA restriction enzyme from H. influenzae Rf232.

A restriction endonuclease has been partially purified from Haemophilus influenzae Rf232 containing the genetically determined system of restriction and modification of DNA. The enzyme requires ATP for the degradation of transfecting phage DNA.     

Utilization of enterobactin and other exogenous iron sources by Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus

The ability of Haemophilus influenzae, H. parainfluenzae and H. paraphrophilus to utilize iron complexes, iron-proteins and exogenous microbial siderophores was evaluated. In a plate bioassay, all three species used not only ferric nitrate but also the iron chelates ferric citrate, ferric nitrilotriacetate and ferric 2,3-dihydroxybenzoate. Each Haemophilus species examined also used haemin, haemoglobin and haem-albumin as iron sources although only H. influenzae could acquire iron from transferrin or from haemoglobin complexed with haptoglobin. None of the haemophili obtained iron from ferritin or lactoferrin or from the microbial siderophores aerobactin or desferrioxamine B. However, the phenolate siderophore enterobactin supplied iron to both H. parainfluenzae and H. paraphrophilus, and DNA isolated from both organisms hybridized with a DNA probe prepared from the Escherichia coli ferric enterobactin receptor gene fepA. In addition, a monospecific polyclonal antiserum raised against the E. coli 81 kDa ferric enterobactin receptor (FepA) recognized an iron-repressible outer membrane protein (OMP) in H. parainfluenzae of between 80 and 82 kDa (depending on the strain). This anti-FepA serum did not cross-react with any of the OMPs of H. paraphrophilus or H. influenzae. The OMPs of each Haemophilus species were also probed with antisera raised against the 74 kDa Cir or 74 kDa IutA (aerobactin receptor) proteins of E. coli. Apart from one H. parainfluenzae strain (NCTC 10665), in which an OMP of about 80 kDa cross-reacted with the anti-IutA sera, no cross-reactivity was observed between Cir, IutA and the OMPs of H. influenzae, H. parainfluenzae or H. paraphrophilus.(ABSTRACT TRUNCATED AT 250 WORDS)     

The molecular mechanism of phase variation of H. influenzae lipopolysaccharide

Multiple carbohydrate structures on the outer-membrane lipopolysaccharide (LPS) of the gram-negative pathogen H. influenzae undergo high frequency, reversible loss, indicative of phase variation. Characterization of a genetic locus, lic-1, responsible for expression of two LPS epitopes displaying phase variation, showed it to comprise four genes. The first gene mediates phase variation. At its 5' end, within the open reading frame, are a variable number of tandem repeats of the tetramer CAAT. By shifting upstream initiation codons in or out of frame, these 4 bp units create a translational switch. The phenotype of organisms corresponds to the number of 4 bp units. Phase variation between three levels of expression ( +, +, and -) of lic-1-derived epitopes is caused by differences in the three phases of translation of the 5' terminus of this gene. Phase variation also allows for selection of organisms displaying certain LPS epitopes in vivo.     

New shuttle vectors for Haemophilus influenzae and Escherichia coli: P15A-derived plasmids replicate in H. influenzae Rd

With the aim of identifying new plasmids useful for molecular cloning in Haemophilus influenzae, several P15A-derived plasmids were tested for their ability to transform H. influenzae Rd. The four plasmids tested, pACYC177, pACYC184, pSU2718, and pSU2719 were all able to establish in H. influenzae Rd. The plasmids were stable, could be purified by standard protocols, and were compatible with a plasmid carrying the RSF0885 origin of replication.