Association of host proteins with the broad host range filamentous phage NgoΦ6 of Neisseria gonorrhoeae.

All Neisseria gonorrhoeae strains contain multiple copies of integrated filamentous phage genomes with undefined structures. In this study, we sought to characterize the capsid proteins of filamentous N. gonorrhoeae bacteriophage NgoΦ6 and phagemids propagated in different bacteria. The data demonstrate that purified phage contain phage-encoded structural proteins and bacterial host proteins; host proteins consistently copurified with the phage particles. The bacterial host proteins associated with the phage filament (as identified by mass spectrometry) tended to be one of the predominant outer membrane components of the host strain, plus minor additional host proteins. We were able to copurify a functional ß-lactamase, a phagemid-encoded protein, with phage filaments. We used protein modeling and immunological analysis to identify the major phage encoded structural proteins. The antigenic properties of these proteins depended on the bacterium where the phages were propagated. Polyclonal antibodies against N. gonorrhoeae phage NgoΦ6 recognized phage-encoded proteins if the phage was propagated in N. gonorrhoeae or H. influenzae cells but not if it was propagated in Salmonella or E. coli. We show that the phage filaments isolated from gonococci and Haemophilus are glycosylated, and this may explain the antigenic diversity seen. Taken en toto, the data demonstrate that while the neisserial filamentous phage are similar to other Inovirus with respect to overall genomic organization, their ability to closely associate with host proteins suggests that they have unique surface properties and are secreted by a here-to-fore unknown secretory pathway.

Haemophilus influenzae HP1 Bacteriophage Encodes a Lytic Cassette with a Pinholin and a Signal-Arrest-Release Endolysin.

HP1 is a temperate bacteriophage, belonging to the Myoviridae family and infecting Haemophilus influenzae Rd. By in silico analysis and molecular cloning, we characterized lys and hol gene products, present in the previously proposed lytic module of HP1 phage. The amino acid sequence of the lys gene product revealed the presence of signal-arrest-release (SAR) and muraminidase domains, characteristic for some endolysins. HP1 endolysin was able to induce lysis on its own when cloned and expressed in Escherichia coli, but the new phage release from infected H. influenzae cells was suppressed by inhibition of the secretion (sec) pathway. Protein encoded by hol gene is a transmembrane protein, with unusual C-out and N-in topology, when overexpressed/activated. Its overexpression in E. coli did not allow the formation of large pores (lack of leakage of ß-galactosidase), but caused cell death (decrease in viable cell count) without lysis (turbidity remained constant). These data suggest that lys gene encodes a SAR-endolysin and that the hol gene product is a pinholin. HP1 SAR-endolysin is responsible for cell lysis and HP1 pinholin seems to regulate the cell lysis and the phage progeny release from H. influenzae cells, as new phage release from the natural host was inhibited by deletion of the hol gene.

Bacterial biofilm in adenoids of children with chronic otitis media. Part II: a case-control study of nasopharyngeal microbiota, virulence, and resistance of biofilms in adenoids.

Background: We previously described that adenoid tissue in children with chronic otitis media (COM) contained more mucosal biofilms than adenoid tissue removed for hypertrophy.Aims/objectives: The aim of the second part was to characterize nasopharyngeal microbiota and explore virulence of the most common middle ear pathogens.Material and methods: Bacteriological analysis was performed following a culture-based approach on the samples recovered from 30 patients of COM group (15 biofilm-positive and 15 biofilm-negative) and from 30 patients of a control group (15 biofilm-positive and 15 biofilm-negative). Virulence factors of Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus influenzae were investigated.Results: The most frequent species were Firmicutes followed by Proteobacteria and Actinobacteria. The presence of biofilm was statistically associated with an increase of the number of bacterial species and Firmicutes phylum regardless of the condition (case/control). No virulence factors associated with invasive isolates were found for the most common middle ear pathogens.Conclusions and significance: This case-control study demonstrated that the presence of COM plus biofilm was associated with a given microbiota which contained more Firmicutes. Our study allows a better understanding of physiopathological mechanisms involved in chronic otitis media and paves the way for further investigations.

Significance of tagI and mfd genes in the virulence of non-typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi) is an opportunist pathogen well adapted to the human upper respiratory tract and responsible for many respiratory diseases. In the human airway, NTHi is exposed to pollutants, such as alkylating agents, that damage its DNA. In this study, we examined the significance of genes involved in the repair of DNA alkylation damage in NTHi virulence. Two knockout mutants, tagI and mfd, encoding N3 methyladenine-DNA glycosylase I and the key protein involved in transcription-coupled repair, respectively, were constructed and their virulence in a BALB/c mice model was examined. This work shows that N3 -methyladenine-DNA glycosylase I is constitutively expressed in NTHi and that it is relevant for its virulence (AU)

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Bacterial pathogens in the nasopharynx, nasal cavity, and osteomeatal complex during wellness and viral infection.

BACKGROUND: Viral sinusitis can precede acute bacterial sinusitis, but the influence of viral infection on bacterial colonization is unclear. The objective of this study was to evaluate the presence of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in the osteomeatal complex (OMC), nasal cavity, and nasopharynx in adults during wellness and viral upper respiratory illness (URI). METHODS: Subjects were recruited for the study during wellness and at the time of acute viral rhinosinusitis. Swab cultures were obtained from the OMC, nasal cavity, and the nasopharynx. Swab eluates were inoculated on selective agars to detect S. pneumoniae, H. influenzae, and M. catarrhalis. RESULTS: The study included 237 subjects, 100 adults with URI and 137 well adults. Positive culture results were found for any site in 70% (n = 70) of ill subjects and 64% (n = 88) of well subjects (p = 0.393). Of the 91 OMC cultures, positive cultures were over five times more likely to be found in ill subjects than in well subjects (31% versus 8%; p = 0.010). The nasal cavity cultures were positively statistically significant more often in ill subjects versus well subjects (39% versus 25%; p = 0.022). The overall nasopharyngeal cultures did not show a statistically significant difference (65% versus 60%; odds ratio, 1.2; p = 0.461). S. pneumoniae was positively cultured in at least one site in 15% of ill subjects and 31% of well subjects (p = 0.006). H. influenzae was positively cultured in at least one site in 45% of ill subjects and 31% of well subjects (p = 0.027). M. catarrhalis was positively cultured in at least one site in 42% of ill subjects and 27% of well subjects (p = 0.018). CONCLUSION: This study defines the carriage rates of the three most common bacterial pathogens for acute sinusitis in the nasopharynx, nasal cavity, and OMC during illness and in the healthy state.

Characterization of the dsDNA prophage sequences in the genome of Neisseria gonorrhoeae and visualization of productive bacteriophage.

BACKGROUND: Bioinformatic analysis of the genome sequence of Neisseria gonorrhoeae revealed the presence of nine probable prophage islands. The distribution, conservation and function of many of these sequences, and their ability to produce bacteriophage particles are unknown. RESULTS: Our analysis of the genomic sequence of FA1090 identified five genomic regions (NgoPhi1 - 5) that are related to dsDNA lysogenic phage. The genetic content of the dsDNA prophage sequences were examined in detail and found to contain blocks of genes encoding for proteins homologous to proteins responsible for phage DNA replication, structural proteins and proteins responsible for phage assembly. The DNA sequences from NgoPhi1, NgoPhi2 and NgoPhi3 contain some significant regions of identity. A unique region of NgoPhi2 showed very high similarity with the Pseudomonas aeruginosa generalized transducing phage F116. Comparative analysis at the nucleotide and protein levels suggests that the sequences of NgoPhi1 and NgoPhi2 encode functionally active phages, while NgoPhi3, NgoPhi4 and NgoPhi5 encode incomplete genomes. Expression of the NgoPhi1 and NgoPhi2 repressors in Escherichia coli inhibit the growth of E. coli and the propagation of phage lambda. The NgoPhi2 repressor was able to inhibit transcription of N. gonorrhoeae genes and Haemophilus influenzae HP1 phage promoters. The holin gene of NgoPhi1 (identical to that encoded by NgoPhi2), when expressed in E. coli, could serve as substitute for the phage lambda s gene. We were able to detect the presence of the DNA derived from NgoPhi1 in the cultures of N. gonorrhoeae. Electron microscopy analysis of culture supernatants revealed the presence of multiple forms of bacteriophage particles. CONCLUSION: These data suggest that the genes similar to dsDNA lysogenic phage present in the gonococcus are generally conserved in this pathogen and that they are able to regulate the expression of other neisserial genes. Since phage particles were only present in culture supernatants after induction with mitomycin C, it indicates that the gonococcus also regulates the expression of bacteriophage genes.

The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection.

Haemophilus influenzae uses phase variation (PV) to modulate the activity of its defence systems against phage infection. The PV of the restriction-modification (R-M) system HindI, the main defence system against phage infection and incoming chromosomal and phage DNA in H. influenzae Rd, is driven by changes of the pentanucleotide repeat tract within the coding sequence of the hsdM gene and is influenced by lack of Dam methylation. Phase-variable resistance/sensitivity to phage infection correlates with changes in lipooligosaccharide (LOS) structure and occurs by slippage of tetranucleotide repeats within the gene lic2A, coding for a step in the biosynthesis of LOS. The lack of Dam activity destabilizes the tetranuclotide (5'-CAAT) repeat tract and increases the frequency of switching from sensitivity to resistance to phage infection more than in the opposite direction. The PV of the lgtC gene does not influence resistance or sensitivity to phage infection. Insertional inactivation of lic2A, but not lgtC or lgtF, leads to resistance to phage infection and to the same structure of the LOS as observed among phase-variable phage-resistant variants. This indicates that in the H. influenzae Rd LOS only the first two sugars (Glc-Gal) extending from the third heptose are part of bacterial phage receptors.

Functional comparison of the transposition core machineries of phage Mu and Haemophilus influenzae Mu-like prophage Hin-Mu reveals interchangeable components.

Bacteriophage Mu uses DNA transposition for propagation and is a model for transposition studies in general. Recent identification of Mu-like prophages within bacterial genomes offers new material for evolutionary and comparative functional studies. One such prophage, Hin-Mu of Haemophilus influenzae Rd, was studied for its transpositional properties. The components of its transposition core machinery, the encoded transposase (MuA(Hin)) and the transposase binding sites, were evaluated for functional properties by sequence comparisons and DNase I footprinting. Transpositional activity of Hin-Mu was examined by in vitro assays directly assessing the assembly and catalytic function of the transposition core machinery. The Hin-Mu components readily assembled catalytically competent protein-DNA complexes, transpososomes. Thus, Hin-Mu encodes a functional transposase and contains critical transposase binding sites. Despite marked sequence differences, components of the Hin-Mu and Mu transposition core machineries are partially interchangeable, reflecting both conservation and flexibility in the functionally important regions within the transpososome structure.

Bacteriophage HP2 of Haemophilus influenzae.

Temperate bacteriophages effect chromosomal evolution of their bacterial hosts, mediating rearrangements and the acquisition of novel genes from other taxa. Although the Haemophilus influenzae genome shows evidence of past phage-mediated lateral transfer, the phages presumed responsible have not been identified. To date, six different H. influenzae phages are known; of these, only the HP1/S2 group, which lyosogenizes exclusively Rd strains (which were originally encapsulated serotype d), is well characterized. Phages in this group are genetically very similar, with a highly conserved set of genes. Because the majority of H. influenzae strains are nonencapsulated (nontypeable), it is important to characterize phages infecting this larger, genetically more diverse group of respiratory pathogens. We have identified and sequenced HP2, a bacteriophage of nontypeable H. influenzae. Although related to the fully sequenced HP1 (and even more so to the partially sequenced S2) and similar in genetic organization, HP2 has a few novel genes and differs in host range; HP2 will not infect or lysogenize Rd strains. Genomic comparisons between HP1/S2 and HP2 suggest recent divergence, with new genes completely replacing old ones at certain loci. Sequence comparisons suggest that H. influenzae phages evolve by recombinational exchange of genes with each other, with cryptic prophages, and with the host chromosome.

Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus.

We report the complete 36,717 bp genome sequence of bacteriophage Mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete Mu-like prophage genomes found in the genomes of a diverse group of bacteria. The comparative studies confirm that members of the Mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups of phages such as the phage lambda-related group of phages of enteric hosts and the phage L5-related group of mycobacteriophages. Mu also possesses segments of similarity, typically gene-sized, to genomes of otherwise non-Mu-like phages. The comparisons show that some well-known features of the Mu genome, including the invertible segment encoding tail fiber sequences, are not present in most members of the Mu genome sequence family examined here, suggesting that their presence may be relatively volatile over evolutionary time. The head and tail-encoding structural genes of Mu have only very weak similarity to the corresponding genes of other well-studied phage types. However, these weak similarities, and in some cases biochemical data, can be used to establish tentative functional assignments for 12 of the head and tail genes. These assignments are strongly supported by the fact that the order of gene functions assigned in this way conforms to the strongly conserved order of head and tail genes established in a wide variety of other phages. We show that the Mu head assembly scaffolding protein is encoded by a gene nested in-frame within the C-terminal half of another gene that encodes the putative head maturation protease. This is reminiscent of the arrangement established for phage lambda.

Site-specific recombination with the chromosomal tRNA(Leu) gene by the large conjugative Haemophilus resistance plasmid.

Characterization of the sequences involved in recombination of the Haemophilus plasmid p1056 with the Haemophilus influenzae chromosome produced evidence indicating site-specific recombination with chromosomal tRNA(Leu). attP sequences identical to those of p1056 were found in six plasmids of diverse origin, suggesting that a family of Haemophilus plasmids recombines with chromosomal tRNA(Leu).

Evidence-based guidelines for treatment of bacterial respiratory tract infections in the era of antibiotic resistance.

Antimicrobial resistance in bacterial respiratory tract pathogens is a rapidly evolving and increasingly disconcerting problem. Major factors that have contributed to resistance are inappropriate prescribing of antibiotics for viral infections and the use of antibiotics with poor activity. The treatment of respiratory tract infections is significantly affected by resistance in organisms such as Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Resistance to beta-lactams, sulfonamides, and macrolides continues to rise. Evidence-based guidelines, founded on clinical and bacteriological outcomes, are imperative to treat patients effectively, to limit the spread of these pathogens, and to minimize further development of resistance. Pharmacokinetic and pharmacodynamic parameters have recently been shown to correlate with clinical outcome, and offer a more rational approach to predicting antimicrobial efficacy and determining clinically relevant susceptibility breakpoints.

Mu-like Prophage in serogroup B Neisseria meningitidis coding for surface-exposed antigens.

Sequence analysis of the genome of Neisseria meningititdis serogroup B revealed the presence of an approximately 35-kb region inserted within a putative gene coding for an ABC-type transporter. The region contains 46 open reading frames, 29 of which are colinear and homologous to the genes of Escherichia coli Mu phage. Two prophages with similar organizations were also found in serogroup A meningococcus, and one was found in Haemophilus influenzae. Early and late phage functions are well preserved in this family of Mu-like prophages. Several regions of atypical nucleotide content were identified. These likely represent genes acquired by horizontal transfer. Three of the acquired genes are shown to code for surface-associated antigens, and the encoded proteins are able to induce bactericidal antibodies.

Cloning of the Dam methyltransferase gene from Haemophilus influenzae bacteriophage HP1.

The putative product of orf13 from the genome of Haemophilus influenzae HP1 bacteriophage shows homology only to bacteriophage T1 Dam methyltransferase, and a weak similarity to the conserved amino acids sequence motifs characteristic of m6A-methyltransferases. Especially interesting is lack of characteristic motif I responsible for binding of S-adenosylmethionine. Despite this fact, a DNA sequence of HP1 bacteriophage of Haemophilus influenzae encoding methyltransferase activity was cloned and expressed in Escherichia coli using pMPMT4 omega expression vector. The cloned methyltransferase recognizes the sequence 5'-GATC-3' and methylates an adenine residue. The enzyme methylates both double- and single-stranded DNA substrates.

Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage.

We report DNA and predicted protein sequence similarities, implying homology, among genes of double-stranded DNA (dsDNA) bacteriophages and prophages spanning a broad phylogenetic range of host bacteria. The sequence matches reported here establish genetic connections, not always direct, among the lambdoid phages of Escherichia coli, phage phiC31 of Streptomyces, phages of Mycobacterium, a previously unrecognized cryptic prophage, phiflu, in the Haemophilus influenzae genome, and two small prophage-like elements, phiRv1 and phiRv2, in the genome of Mycobacterium tuberculosis. The results imply that these phage genes, and very possibly all of the dsDNA tailed phages, share common ancestry. We propose a model for the genetic structure and dynamics of the global phage population in which all dsDNA phage genomes are mosaics with access, by horizontal exchange, to a large common genetic pool but in which access to the gene pool is not uniform for all phage.

Identification of the second attachment site for HP1 and S2 bacteriophages in Haemophilus influenzae genome.

Presented data demonstrate that both HP1c1 and S2 bacteriophages of Haemophilus influenzae can use two related attB sites on the host chromosome. The first indication comes from the analysis of the whole genome sequence of H. influenzae Rd in which a second region of nucleotide sequence identical with the known attB site was detected. The location of both attB sites agrees with results of earlier field-inversion gel electrophoresis (FIGE) analysis of S2 and HP1c1 lysogens of H. influenzae. Functionality of cloned attB sites in the integration process of both phages was confirmed using in vivo integrase-dependent recombination system in Escherichia coli. Presented results seem to extend previously described similarities between S2 and HP1c1 phages.

The relationship between HP1 and S2 bacteriophages of Haemophilus influenzae.

Comparison of the nucleotide sequences of the left arms of two Haemophilus influenzae phages, S2 and HP1 is presented. They exhibit a characteristic mosaic pattern of homologous and non-homologous regions. The homology extends over the attP site and int, orf 5 to 9, rep and the 3' part of cI genes. Two major non-homologous regions were detected. One is found between the int and cI genes; the other spans the region of promoters and the cox gene. Variations in the region of the promotors which is involved in the choice between a lysogenic and a lytic pathway and some divergences in the cI coding sequences are probably responsible for the observed immunity differences between the two phages. Distinctions in the distribution of consensus sequences for an integration host factor (IHF) and integrase-binding sites and promoters are described. These data offer an explanation of the relationship between three types of S2/HP1 phages. It allows in turn a final settlement of the nomenclature variation in the literature. The results presented, which are similar to those obtained for other phage groups, suggest that the mosaic structure of phage genomes is a normal outcome of phage divergence.

Reciprocal regulation of the early promoter region of bacteriophage HP1 by the Cox and Cl proteins.

We have identified a transcriptional switch at the early promoter region of bacteriophage HP1. This switch controls the transcription of the early lytic operon from the P(R1) and P(R2) promoters and the transcription of the lysogenic operon from the P(L) promoter. The start sites of the three promoters were mapped, and using a chloramphenicol acetyl transferase assay, we have investigated the levels of transcription from the promoters in the absence or in the presence of two phage-encoded transcription factors: HP1 Cox and HP1 Cl. The HP1 Cox protein repressed the production of P(L) transcripts 30-fold, while the HP1 Cl protein repressed lytic transcription at least 70-fold. Binding sites for HP1 Cox and Cl were identified in the early promoter region; mutations of these sites eliminated transcriptional repression. In addition, a mutant Cl protein was isolated which is temperature sensitive for repression. Taken together, these data demonstrate the reciprocal regulation of a transcriptional switch in which the actions of the two phage-encoded proteins at the phage early promoters determine the choice between lytic and lysogenic growth.

Purification and characterization of HP1 Cox and definition of its role in controlling the direction of site-specific recombination.

The protein that activates site-specific excision of the HP1 genome from the Hemophilus influenzae chromosome, HP1 Cox, was purified. Native Cox consists of four 8.9-kDa protomers. Tetrameric Cox self-associates to octamers; the apparent dissociation constant was 8 microM protomer, suggesting that under reaction conditions Cox is largely tetrameric. Cox binding sites consist of two direct repeats of the consensus motif 5'-GGTMAWWWWA; one Cox tetramer binds to each motif. Cox binding interfered with the interaction of HP1 integrase with one of its binding sites, IBS5. This competition is central to directional control, as shown by studies on mutated sites. Both Cox binding sites were necessary for Cox to fully inhibit integration and activate excision, although Cox continued to affect recombination when the single binding site proximal to IBS5 remained intact. Eliminating the IBS5 site completely prevented integration but greatly enhanced excision. Excisive recombination continued to require Cox even when IBS5 was inactivated. Cox must therefore play a positive role in assembling the nucleoprotein complexes producing excisive recombination, by inducing the formation of a critical conformation in those complexes.