Identification of a siderophore utilization locus in nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis>

Background  

Haemophilus influenzae has an absolute aerobic growth requirement for either heme, or iron in the presence of protoporphyrin IX. Both iron and heme in the mammalian host are strictly limited in their availability to invading microorganisms. Many bacterial species overcome iron limitation in their environment by the synthesis and secretion of small iron binding molecules termed siderophores, which bind iron and deliver it into the bacterial cell via specific siderophore receptor proteins on the bacterial cell surface. There are currently no reports of siderophore production or utilization by H. influenzae.     

<Emphasis Type="Italic">In vitro</Emphasis> activity of telithromycin against <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> at epithelial lining fluid concentrations

Background  

Haemophilus influenzae is one of the main aetiological agents of community-acquired respiratory tract infections. The primary aim of this study was to evaluate the antibacterial activity of telithromycin against H. influenzae clinical isolates showing different pattern of resistance in comparison with azithromycin and clarithromycin at 1/4 ×, 1/2 ×, 1 ×, 2 ×, 4 × minimum inhibitory concentration (MIC) and to peak concentrations in epithelial lining fluid (ELF). The secondary aim was to determine the influence of CO₂ enriched atmosphere on bacterial susceptibility.     

DNA-binding by Haemophilus influenzae and Escherichia coli YbaB, members of a widely-distributed bacterial protein family

Background  

Genes orthologous to the ybaB loci of Escherichia coli and Haemophilus influenzae are widely distributed among eubacteria. Several years ago, the three-dimensional structures of the YbaB orthologs of both E. coli and H. influenzae were determined, revealing a novel "tweezer"-like structure. However, a function for YbaB had remained elusive, with an early study of the H. influenzae ortholog failing to detect DNA-binding activity. Our group recently determined that the Borrelia burgdorferi YbaB ortholog, EbfC, is a DNA-binding protein. To reconcile those results, we assessed the abilities of both the H. influenzae and E. coli YbaB proteins to bind DNA to which B. burgdorferi EbfC can bind.     

Identification of biofilm proteins in non-typeable Haemophilus Influenzae

Background  

Non-typeable Haemophilus influenzae biofilm formation is implicated in a number of chronic infections including otitis media, sinusitis and bronchitis. Biofilm structure includes cells and secreted extracellular matrix that is "slimy" and believed to contribute to the antibiotic resistant properties of biofilm bacteria. Components of biofilm extracellular matrix are largely unknown. In order to identify such biofilm proteins an ex-vivo biofilm of a non-typeable Haemophilus influenzae isolate, originally from an otitis media patent, was produced by on-filter growth. Extracellular matrix fraction was subjected to proteomic analysis via LC-MS/MS to identify proteins.     

Prevalence of genetic differences in phosphorylcholine expression between nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> and <Emphasis Type="Italic">Haemophilus haemolyticus</Emphasis>

Background  

Although non-typeable (NT) Haemophilus influenzae and Haemophilus haemolyticus are closely related human commensals, H. haemolyticus is non-pathogenic while NT H. influenzae is an important cause of respiratory tract infections. Phase-variable phosphorylcholine (ChoP) modification of lipooligosaccharide (LOS) is a NT H. influenzae virulence factor that, paradoxically, may also promote complement activation by binding C-reactive protein (CRP). CRP is known to bind more to ChoP positioned distally than proximally in LOS, and the position of ChoP within LOS is dictated by specific licD alleles (designated here as licD _I , licD _III , and licD _IV ) that are present in a lic1 locus. The lic1 locus contains the licA-licD genes, and ChoP-host interactions may also be influenced by a second lic1 locus that allows for dual ChoP substitutions in the same strain, or by the number of licA gene tetranucleotide repeats (5'-CAAT-3') that reflect phase-variation mutation rates.     

Multiplex quantitative PCR for detection of lower respiratory tract infection and meningitis caused by <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis>, <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> and <Emphasis Type="Italic">Neisseria meningitidis</Emphasis>

Background  

Streptococcus pneumoniae and Haemophilus influenzae cause pneumonia and as Neisseria meningitidis they are important agents of meningitis. Although several PCR methods have been described for these bacteria the specificity is an underestimated problem. Here we present a quantitative multiplex real-time PCR (qmPCR) for detection of S. pneumoniae (9802 gene fragment), H. influenzae (omp P6 gene) and N. meningitidis (ctrA gene). The method was evaluated on bronchoalveolar lavage (BAL) samples from 156 adults with lower respiratory tract infection (LRTI) and 31 controls, and on 87 cerebrospinal fluid (CSF) samples from meningitis patients.     

Nontypeable Haemophilus influenzae Genetic Islands Associated with Chronic Pulmonary Infection

Background

Haemophilus influenzae (Hi) colonizes the human respiratory tract and is an important pathogen associated with chronic obstructive pulmonary disease (COPD). Bacterial factors that interact with the human host may be important in the pathogenesis of COPD. These factors, however, have not been well defined. The overall goal of this study was to identify bacterial genetic elements with increased prevalence among H. influenzae strains isolated from patients with COPD compared to those isolated from the pharynges of healthy individuals.

Methodology/Principal Findings

Four nontypeable H. influenzae (NTHi) strains, two isolated from the airways of patients with COPD and two from a healthy individual, were subjected to whole genome sequencing using 454 FLX Titanium technology. COPD strain-specific genetic islands greater than 500 bp in size were identified by in silico subtraction. Open reading frames residing within these islands include known Hi virulence genes such as lic2b, hgbA, iga, hmw1 and hmw2, as well as genes encoding urease and other enzymes involving metabolic pathways. The distributions of seven selected genetic islands were assessed among a panel of 421 NTHi strains of both disease and commensal origins using a Library-on-a-Slide high throughput dot blot DNA hybridization procedure. Four of the seven islands screened, containing genes that encode a methyltransferase, a dehydrogenase, a urease synthesis enzyme, and a set of unknown short ORFs, respectively, were more prevalent in COPD strains than in colonizing strains with prevalence ratios ranging from 1.21 to 2.85 (p≤0.0002). Surprisingly, none of these sequences show increased prevalence among NTHi isolated from the airways of patients with cystic fibrosis.

Conclusions/Significance

Our data suggest that specific bacterial genes, many involved in metabolic functions, are associated with the ability of NTHi strains to survive in the lower airways of patients with COPD.     

Evolution of competence and DNA uptake specificity in the Pasteurellaceae

Background  

Many bacteria can take up DNA, but the evolutionary history and function of natural competence and transformation remain obscure. The sporadic distribution of competence suggests it is frequently lost and/or gained, but this has not been examined in an explicitly phylogenetic context. Additional insight may come from the sequence specificity of uptake by species such as Haemophilus influenzae, where a 9 bp uptake signal sequence (USS) repeat is both highly overrepresented in the genome and needed for efficient DNA uptake. We used the distribution of competence genes and DNA uptake specificity in H. influenzae 's family, the Pasteurellaceae, to examine the ancestry of competence.     

Identification of a second family of high-molecular-weight adhesion proteins expressed by non-typable Haemophilus influenzae

We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of non-typable Haemophilus influenzae (NTHI), mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to 70–75% of NTHI strains. NTHI strains that lack HMW1/ HMW2-like proteins remain capable of efficient attachment to cultured human epithelial cells, suggesting the existence of additional adhesion molecules. We reasoned that characterization of high-molecular-weight immunogenic proteins from an HMW1/HMW2-deficient strain might identify additional adhesion proteins. A genomic library was prepared in λEMBL3 with chromosomal DNA from non-typable Haemophilus strain 11, a strain that lacks HMW1/HMW2-like proteins. The library was screened immunologically with convalescent serum from a child naturally infected with strain 11, and phage clones expressing high-molecular-weight recombinant proteins were identified by Western blot analysis. One clone was identified that expressed a protein with an apparent molecular mass greater than 200 kDa. Transformation of non-adherent Escherichia coli strain DH5α with plasmids containing the genetic locus encoding this protein gave rise to E. colitransformants that adhered avidly to Chang conjunctival cells. Subcloning and mutagenesis studies localized the DNA conferring the adherence phenotype to a 4.8 kbp fragment, and nucleotide sequence analysis further localized the gene encoding the adhesion protein to a 3.3 kbp open reading frame predicted to encode a protein of 114kDa. The gene was designated hia for Haemophilus influenzae adhesin. Southern analysis revealed an hia homologue in 13 of 15 HMW1/HMW2-deficient non-typable H. influenzae strains. In contrast, the hia gene was not present in any of 23 non-typable H. influenzae strains which expressed HMW1/HMW2-like proteins. Identification of this second family of high-molecular-weight adhesion proteins suggests the possibility of developing vaccines based upon a combination of HMW1/HMW2-like proteins and Hia-like proteins which would be protective against disease caused by most or all non-typable H. influenzae     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Sialic acid mediated transcriptional modulation of a highly conserved sialometabolism gene cluster in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> and its effect on virulence

Background  

Sialic acid has been shown to be a major virulence determinant in the pathogenesis of otitis media caused by the bacterium Haemophilus influenzae. This study aimed to characterise the expression of genes required for the metabolism of sialic acid and to investigate the role of these genes in virulence.     

Construction of non-polar mutants in <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> using FLP recombinase technology

Background  

Nontypeable Haemophilus influenzae (NTHi) is a gram-negative bacterium that causes otitis media in children as well as other infections of the upper and lower respiratory tract in children and adults. We are employing genetic strategies to identify and characterize virulence determinants in NTHi. NTHi is naturally competent for transformation and thus construction of most mutants by common methodologies is relatively straightforward. However, new methodology was required in order to construct unmarked non-polar mutations in poorly expressed genes whose products are required for transformation. We have adapted the lambda red/FLP-recombinase-mediated strategy used in E. coli for use in NTHi.     

Identification and characterization of a nontypeable <Emphasis Type="Italic">Haemophilus influenzae</Emphasis> putative toxin-antitoxin locus

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 HI0450). Both vapD _Hi and a flanking gene, HI0451, exhibit the genetic and physical characteristics of a toxin/antitoxin (TA) locus, with VapD _Hi serving as the toxin moiety and HI0451 as the antitoxin. We propose the name VapX _Hi for 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.     

Presence of gastric Helicobacter species in children suffering from gastric disorders in Southern Turkey

Background

Infections with gastric Helicobacter spp. are associated with gastritis, peptic ulceration, and malignancies. Helicobacter pylori is the most prevalent Helicobacter species colonizing the human stomach. Other gastric non‐H. pylori helicobacters (NHPHs) have been described in 0.2%‐6% of human patients with gastric disorders. Nevertheless, due to difficulties in the diagnosis of NHPH infections and lack of routine screening, this is most likely an underestimation of their true prevalence. To the best of our knowledge, no studies have been performed in the presence of Helicobacter spp. in children suffering from gastric disorders in Southern Turkey.

Materials and methods

In total, 110 children with gastric complaints were examined at the Cukurova University Balcali hospital, Turkey. Gastroscopy was performed to evaluate the presence of gastric mucosal lesions. Biopsies of the pyloric gland zone were taken for histopathological analysis, rapid urease testing, and presence of Helicobacter spp. DNA by PCR.

Results

Based on the PCR results, the prevalence of Helicobacter spp. was 32.7% (36/110). H. pylori was found in 30.9% (34/110), H. suis in 1.8% (2/110), and H. heilmannii/H. ailurogastricus in 0.9% (1/110) of the human patients. A mixed infection with H. pylori and H. suis was present in one patient. The presence of mucosal abnormalities, such as nodular inflammation, ulceration, and hyperemia, as well as gastritis, was significantly higher in Helicobacter spp. positive patients.

Conclusion

Helicobacter pylori, H. suis, and H. heilmannii/H. ailurogastricus were present in children with gastric complaints. Infection with these pathogens may be involved in the development of gastritis and ulceration.     

Definition of novel cell envelope associated proteins in Triton X-114 extracts of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> H37Rv

Background  

Membrane- and membrane-associated proteins are important for the pathogenicity of bacteria. We have analysed the content of these proteins in virulent Mycobacterium tuberculosis H37Rv using Triton X-114 detergent-phase separation for extraction of lipophilic proteins, followed by their identification with high resolution mass spectrometry.     

Haemophilus influenzae: using comparative genomics to accurately identify a highly recombinogenic human pathogen

Background

Haemophilus influenzae is an opportunistic bacterial pathogen that exclusively colonises humans and is associated with both acute and chronic disease. Despite its clinical significance, accurate identification of H. influenzae is a non-trivial endeavour. H. haemolyticus can be misidentified as H. influenzae from clinical specimens using selective culturing methods, reflecting both the shared environmental niche and phenotypic similarities of these species. On the molecular level, frequent genetic exchange amongst Haemophilus spp. has confounded accurate identification of H. influenzae, leading to both false-positive and false-negative results with existing speciation assays.

Results

Whole-genome single-nucleotide polymorphism data from 246 closely related global Haemophilus isolates, including 107 Australian isolate genomes generated in this study, were used to construct a whole-genome phylogeny. Based on this phylogeny, H. influenzae could be differentiated from closely related species. Next, a H. influenzae-specific locus, fucP, was identified, and a novel TaqMan real-time PCR assay targeting fucP was designed. PCR specificity screening across a panel of clinically relevant species, coupled with in silico analysis of all species within the order Pasteurellales, demonstrated that the fucP assay was 100 % specific for H. influenzae; all other examined species failed to amplify.

Conclusions

This study is the first of its kind to use large-scale comparative genomic analysis of Haemophilus spp. to accurately delineate H. influenzae and to identify a species-specific molecular signature for this species. The fucP assay outperforms existing H. influenzae targets, most of which were identified prior to the next-generation genomics era and thus lack validation across a large number of Haemophilus spp. We recommend use of the fucP assay in clinical and research laboratories for the most accurate detection and diagnosis of H. influenzae infection and colonisation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1857-x) contains supplementary material, which is available to authorized users.     

CEACAM1 recognition by bacterial pathogens is species-specific

Background  

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), an immunoglobulin (Ig)-related glycoprotein, serves as cellular receptor for a variety of Gram-negative bacterial pathogens associated with the human mucosa. In particular, Neisseria gonorrhoeae, N. meningitidis, Moraxella catarrhalis, and Haemophilus influenzae possess well-characterized CEACAM1-binding adhesins. CEACAM1 is typically involved in cell-cell attachment, epithelial differentiation, neovascularisation and regulation of T-cell proliferation, and is one of the few CEACAM family members with homologues in different mammalian lineages. However, it is unknown whether bacterial adhesins of human pathogens can recognize CEACAM1 orthologues from other mammals.     

Surface display of Salmonella epitopes in <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus carnosus</Emphasis>

Background  

Salmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes.