The role of the RNA chaperone Hfq in Haemophilus influenzae pathogenesis

BMC Microbiology

Terrence L Stull²  Paul W Whitby¹  Thomas W Seale¹  Daniel J Morton¹  Randy J Hempel² 

[1] Department of Pediatrics, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA;Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA

关键词: Bacteremia;    Otitis media;    Pathogenesis;    Hfq;    Haemophilus;   

Others  :  1143623
DOI  :  10.1186/1471-2180-13-134

 received in 2013-03-28, accepted in 2013-06-14,  发布年份 2013

【 摘 要 】

Background

The RNA binding protein Hfq of Haemophilus influenzae is highly homologous to Hfq from other bacterial species. In many of these other bacteria, Hfq affects the expression of a broad range of genes and enhances the ability to respond to stressful environments. However, the role of Hfq in H. influenzae is unknown.

Results

Deletion mutants of hfq were generated in the nontypeable H. influenzae strains R2866 and 86-028NP to assess the role of Hfq in these well characterized but genotypically and phenotypically divergent clinical isolates. A deletion mutation of hfq had no effect on growth of H. influenzae in nutrient rich media and had no effect on survival in several stressful conditions in vitro. However, the mutation resulted in a reduced ability to utilize heme from hemoglobin. The mutant and wild type strains were assessed for virulence and competitive fitness in models of invasive disease and otitis media. In the chinchilla model of otitis media, the hfq mutant of 86-028NP exhibited impaired competitive fitness when compared to its wild type progenitor but exhibited no apparent defect in virulence. In the infant rat model, deletion of hfq in R2866 resulted in reduced bacterial titers in blood and a shorter duration of infection when compared to the wild type strain in the competitive fitness study.

Conclusion

We conclude that Hfq is involved in the utilization of essential nutrients and facilitates infection by H. influenzae.

【 授权许可】

   
2013 Hempel et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

【 参考文献 】

• [1]Turk DC: The pathogenicity of Haemophilus influenzae. J Med Microbiol 1984, 18:1-16.
• [2]García-Rodríguez JÁ, Fresnadillo Martínez MJ: Dynamics of nasopharyngeal colonization by potential respiratory pathogens. J Antimicrob Chemother 2002, 50:59-74.
• [3]Bajanca P, Canica M: Emergence of nonencapsulated and encapsulated non-b-type invasive Haemophilus influenzae isolates in Portugal (1989–2001). J Clin Microbiol 2004, 42:807-810.
• [4]Teele DW, Klein JO, Rosner B: Epidemiology of otitis media during the first seven years of life in children in greater Boston: a prospective, cohort study. J Infect Dis 1989, 160:83-94.
• [5]Evans NM, Smith DD, Wicken AJ: Haemin and nicotinamide adenine dinucleotide requirements of Haemophilus influenzae and Haemophilus parainfluenzae. J Med Microbiol 1974, 7:359-365.
• [6]Herbert M, Kraiss A, Hilpert AK, Schlor S, Reidl J: Aerobic growth deficient Haemophilus influenzae mutants are non-virulent: implications on metabolism. Int J Med Microbiol 2003, 293:145-152.
• [7]Genco CA, Dixon DW: Emerging strategies in microbial haem capture. Mol Microbiol 2001, 39:1-11.
• [8]Schaible UE, Kaufmann SH: Iron and microbial infection. Nat Rev Microbiol 2004, 2:946-953.
• [9]Morton D, Stull T: Haemophilus. In Iron Transport in Bacteria. Edited by Crosa JH, Mey AR, Payne SM. Washington, D.C: American Society for Microbiology; 2004:273-292.
• [10]Whitby PW, Seale TW, VanWagoner TM, Morton DJ, Stull TL: The iron/heme regulated genes of Haemophilus influenzae: comparative transcriptional profiling as a tool to define the species core modulon. BMC Genomics 2009, 10:6. BioMed Central Full Text
• [11]Whitby PW, Vanwagoner TM, Seale TW, Morton DJ, Stull TL: Transcriptional profile of Haemophilus influenzae: effects of iron and heme. J Bacteriol 2006, 188:5640-5645.
• [12]Fantappie L, Metruccio MM, Seib KL, Oriente F, Cartocci E, Ferlicca F, Giuliani MM, Scarlato V, Delany I: The RNA chaperone Hfq is involved in the stress response and virulence in Neisseria meningitidis and is a pleiotropic regulator of protein expression. Infect Immun 2009, 77:1842-1880.
• [13]Kulesus RR, Diaz-Perez K, Slechta ES, Eto DS, Mulvey MA: Impact of the RNA chaperone Hfq on the fitness and virulence potential of uropathogenic Escherichia coli. Infect Immun 2008, 76:3019-3026.
• [14]Sittka A, Pfeiffer V, Tedin K, Vogel J: The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium. Mol Microbiol 2007, 63:193-217.
• [15]de Fernandez MT F, Eoyang L, August JT: Factor fraction required for the synthesis of bacteriophage Qbeta-RNA. Nature 1968, 219:588-590.
• [16]Moller T, Franch T, Hojrup P, Keene DR, Bachinger HP, Brennan RG, Valentin-Hansen P: Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction. Mol Cell 2002, 9:23-30.
• [17]Hajnsdorf E, Boni IV: Multiple activities of RNA-binding proteins S1 and Hfq. Biochimie 2012, 94:1544-1553.
• [18]Masse E, Gottesman S: A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli. Proc Natl Acad Sci U S A 2002, 99:4620-4625.
• [19]Masse E, Salvail H, Desnoyers G, Arguin M: Small RNAs controlling iron metabolism. Curr Opin Microbiol 2007, 10:140-145.
• [20]Mellin JR, McClure R, Lopez D, Green O, Reinhard B, Genco C: Role of Hfq in Iron Dependent and Independent Gene Regulation in Neisseria meningitidis. Microbiology 2010, 156:2316-2326.
• [21]Chao Y, Vogel J: The role of Hfq in bacterial pathogens. Curr Opin Microbiol 2010, 13:24-33.
• [22]Nizet V, Colina KF, Almquist JR, Rubens CE, Smith AL: A virulent nonencapsulated Haemophilus influenzae. J Infect Dis 1996, 173:180-186.
• [23]Bakaletz LO, Kennedy B-J, Novotny LA, Duquesne G, Cohen J, Lobet Y: Protection against Development of Otitis Media Induced by Nontypeable Haemophilus influenzae by Both Active and Passive Immunization in a Chinchilla Model of Virus-Bacterium Superinfection. Infect Immun 1999, 67:2746-2762.
• [24]Morton DJ, Seale TW, Bakaletz LO, Jurcisek JA, Smith A, VanWagoner TM, Whitby PW, Stull TL: The heme-binding protein (HbpA) of Haemophilus influenzae as a virulence determinant. Int J Med Microbiol 2009, 299:479-488.
• [25]Poje G, Redfield RJ: General methods for culturing Haemophilus influenzae. Methods Mol Med 2003, 71:51-56.
• [26]Whitby PW, Morton DJ, Stull TL: Construction of antibiotic resistance cassettes with multiple paired restriction sites for insertional mutagenesis of Haemophilus influenzae. FEMS Microbiol Lett 1998, 158:57-60.
• [27]Morton DJ, Smith A, Ren Z, Madore LL, VanWagoner TM, Seale TW, Whitby PW, Stull TL: Identification of a haem-utilization protein (Hup) in Haemophilus influenzae. Microbiology 2004, 150:3923-3933.
• [28]Saeed-Kothe A, Yang W, Mills SD: Use of the Riboflavin Synthase Gene (ribC) as a Model for Development of an Essential Gene Disruption and Complementation System for Haemophilus influenzae. Appl Environ Microbiol 2004, 70:4136-4143.
• [29]Whitby PW, Morton DJ, Vanwagoner TM, Seale TW, Cole BK, Mussa HJ, McGhee PA, Bauer CY, Springer JM, Stull TL: Haemophilus influenzae OxyR: characterization of its regulation, regulon and role in fitness. PLoS One 2012, 7:e50588.
• [30]Whitby PW, Seale TW, Morton DJ, VanWagoner TM, Stull TL: Characterization of the Haemophilus influenzae tehB gene and its role in virulence. Microbiology 2010, 156:1188-1200.
• [31]Munson R Jr, Hunt A: Isolation and characterization of a mutant of Haemophilus influenzae type b deficient in outer membrane protein P1. Infect Immun 1989, 57:1002-1004.
• [32]Segada LM, Carlone GM, Gheesling LL, Lesse AJ: Characterization of P1-deficient isogenic mutant of Haemophilus influenzae biogroup aegyptius associated with Brazilian purpuric fever. Microb Pathog 2000, 28:145-155.
• [33]Bolduc GR, Bouchet V, Jiang RZ, Geisselsoder J, Truong-Bolduc QC, Rice PA, Pelton SI, Goldstein R: Variability of outer membrane protein P1 and its evaluation as a vaccine candidate against experimental otitis media due to nontypeable Haemophilus influenzae: an unambiguous, multifaceted approach. Infect Immun 2000, 68:4505-4517.
• [34]Jorth P, Whiteley M: Characterization of a novel riboswitch-regulated lysine transporter in Aggregatibacter actinomycetemcomitans. J Bacteriol 2010, 192:6240-6250.
• [35]Lloyd AL, Marshall BJ, Mee BJ: Identifying cloned Helicobacter pylori promoters by primer extension using a FAM-labelled primer and GeneScan® analysis. J Microbiol Methods 2005, 60:291-298.
• [36]Morton DJ, Madore LL, Smith A, Vanwagoner TM, Seale TW, Whitby PW, Stull TL: The heme-binding lipoprotein (HbpA) of Haemophilus influenzae: role in heme utilization. FEMS Microbiol Lett 2005, 253:193-199.
• [37]Morton DJ, VanWagoner TM, Seale TW, Whitby PW, Stull TL: Differential utilization by Haemophilus influenzae of haemoglobin complexed to the three human haptoglobin phenotypes. FEMS Immunol Med Microbiol 2006, 46:426-432.
• [38]Jett BD, Hatter KL, Huycke MM, Gilmore MS: Simplified agar plate method for quantifying viable bacteria. Biotechniques 1997, 23:648-650.
• [39]Bakaletz LO, Leake ER, Billy JM, Kaumaya PT: Relative immunogenicity and efficacy of two synthetic chimeric peptides of fimbrin as vaccinogens against nasopharyngeal colonization by nontypeable Haemophilus influenzae in the chinchilla. Vaccine 1997, 15:955-961.
• [40]Gitiban N, Jurcisek JA, Harris RH, Mertz SE, Durbin RK, Bakaletz LO, Durbin JE: Chinchilla and murine models of upper respiratory tract infections with respiratory syncytial virus. J Virol 2005, 79:6035-6042.
• [41]Morton DJ, Bakaletz LO, Jurcisek JA, VanWagoner TM, Seale TW, Whitby PW, Stull TL: Reduced severity of middle ear infection caused by nontypeable Haemophilus influenzae lacking the hemoglobin/hemoglobin-haptoglobin binding proteins (Hgp) in a chinchilla model of otitis media. Microb Pathog 2004, 36:25-33.
• [42]Smith AL, Smith DH, Averill DR Jr, Marino J, Moxon ER: Production of Haemophilus influenzae b meningitis in infant rats by intraperitoneal inoculation. Infect Immun 1973, 8:278-290.
• [43]Seale TW, Morton DJ, Whitby PW, Wolf R, Kosanke SD, VanWagoner TM, Stull TL: Complex Role of Hemoglobin and Hemoglobin-Haptoglobin Binding Proteins in Haemophilus influenzae Virulence in the Infant Rat Model of Invasive Infection. Infect Immun 2006, 74:6213-6225.
• [44]Nielsen JS, Boggild A, Andersen CB, Nielsen G, Boysen A, Brodersen DE, Valentin-Hansen P: An Hfq-like protein in archaea: crystal structure and functional characterization of the Sm protein from Methanococcus jannaschii. RNA 2007, 13:2213-2223.
• [45]Olsen AS, Møller-Jensen J, Brennan RG, Valentin-Hansen P: C-Terminally Truncated Derivatives of Escherichia coli Hfq Are Proficient in Riboregulation. J Mol Biol 2010, 404:173-182.
• [46]Morton DJ, Hempel RJ, Seale TW, Whitby PW, Stull TL: A functional tonB gene is required for both virulence and competitive fitness in a chinchilla model of Haemophilus influenzae otitis media. BMC Res Notes 2012, 5:327. BioMed Central Full Text
• [47]Tsao D, Nelson KL, Kim D, Smith AL: Infant rat infection modifies phenotypic properties of an invasive nontypeable Haemophilus influenzae. Microbes Infect 2012, 14:509-516.
• [48]Mason KM, Munson RS Jr, Bakaletz LO: A mutation in the sap operon attenuates survival of nontypeable Haemophilus influenzae in a chinchilla model of otitis media. Infect Immun 2005, 73:599-608.
• [49]St Geme JW 3rd: Molecular and cellular determinants of non-typeable Haemophilus influenzae adherence and invasion. Cell Microbiol 2002, 4:191-200.
• [50]Wilcox KW, Smith HO: Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5′-triphosphate-dependent deoxyribonuclease activity. J Bacteriol 1975, 122:443-453.
• [51]Chambers JR, Bender KS: The RNA Chaperone Hfq Is Important for Growth and Stress Tolerance in Francisella novicida. PLoS One 2011, 6:e19797.
• [52]Tsui HC, Leung HC, Winkler ME: Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol 1994, 13:35-49.
• [53]Sousa SA, Ramos CG, Moreira LM, Leitao JH: The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans. Microbiology 2010, 156:896-908.
• [54]Vecerek B, Moll I, Afonyushkin T, Kaberdin V, Blasi U: Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli. Mol Microbiol 2003, 50:897-909.
• [55]Meibom KL, Forslund AL, Kuoppa K, Alkhuder K, Dubail I, Dupuis M, Forsberg A, Charbit A: Hfq, a novel pleiotropic regulator of virulence-associated genes in Francisella tularensis. Infect Immun 2009, 77:1866-1880.
• [56]Geng J, Song Y, Yang L, Feng Y, Qiu Y, Li G, Guo J, Bi Y, Qu Y, Wang W, et al.: Involvement of the Post-Transcriptional Regulator Hfq in Yersinia pestis Virulence. PLoS One 2009, 4:e6213.
• [57]Morton DJ, Whitby PW, Jin H, Ren Z, Stull TL: Effect of multiple mutations in the hemoglobin- and hemoglobin-haptoglobin-binding proteins, HgpA, HgpB, and HgpC, of Haemophilus influenzae type b. Infect Immun 1999, 67:2729-2739.
• [58]Mann B, van Opijnen T, Wang J, Obert C, Wang Y-D, Carter R, McGoldrick DJ, Ridout G, Camilli A, Tuomanen EI, Rosch JW: Control of Virulence by Small RNAs in Streptococcus pneumoniae. PLoS Pathog 2012, 8:e1002788.
• [59]Ding Y, Davis BM, Waldor MK: Hfq is essential for Vibrio cholerae virulence and downregulates sigma expression. Mol Microbiol 2004, 53:345-354.