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Exploring Haemophilus haemolyticus by functional genomic analyses

Exploring Haemophilus haemolyticus by functional genomic analyses. Xin Wang, Ph. D Meningitis Laboratory, CDC. MVPDB/DBD/NCIRD WHO Collaborating Center. Taxonomic characteristics of Haemophilus genus of the family Pasteurellaceae. Small, gram-negative coccobacilli

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Exploring Haemophilus haemolyticus by functional genomic analyses

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  1. Exploring Haemophilus haemolyticus by functional genomic analyses Xin Wang, Ph. D Meningitis Laboratory, CDC MVPDB/DBD/NCIRD WHO Collaborating Center

  2. Taxonomic characteristics of Haemophilus genus of the family Pasteurellaceae • Small, gram-negative coccobacilli • Positive for cytochromeoxidase • Growth in culture requires exogenous hemin (oxidized ferroprotoporphyrin) (X factor) and/or nicotinamide adenine dinucleotide (NAD) (V factor) Haemophilus influenzae

  3. Common Haemophilus Species • Haemophilus haemolyticus (Hh) • Haemophilus influenzae (Hi) • Haemophilus parainfluenzae • Haemophilus parahaemolyticus • Haemophilus paraphrohaemolyticus • Haemophilus aphrophilus(Reclassified) • Haemophilus paraphrophilus (Reclassified) • Haemophilus segnis(Reclassified) • Haemophilus ducreyi(Hd) Human commensal

  4. HhvsNTHi • Hh shares high similarity with Hi: • Colony and cell morphology • Carbohydrate metabolism • Biochemical features • Genetic background • Doesn’t react with Hi a-f antiserum • Discriminating NTHi from Hh • Beta-hemolysis. • Loss of hemolysis in some Hh strains. • Often misidentified as NTHi. • Gene(s) encoding hemolysin unknown

  5. Identification of Hi and Hh • Colony morphology on selective media • Kovac’soxidase test to determine • the presence of cytochromeoxidase • Porphyrin test : exclude • hemin-independent Haemophilus strains • Hemo ID QUAD plates to NAD and • hemin-dependent growth • API NH strips to determine the identity of the selected colonies • PCR to detect biomaker genes (fucK, hpd, iga, lgtC) • 16S rRNA gene sequencing (on selected isolates)

  6. A new simple typing scheme is needed!

  7. Timeline for performing different typing methods Phenotype-based assays: a few hours to a day Genotype-based assays PCR: hours gene sequencing: days Genome-based assays: days or weeks

  8. Hhvs Hi Hi and Hhare most prevalent human commensal and also cause disease. Encapsulated Hi serotypes a-f: -invasive infection: pneumonia, meningitis, and bacteremia -person-to-person spread due to inhalation of infectious droplets -type b Hiwas most commonly prior to Hib conjugate vaccine -prevalence of Hib cases has declined with advent of vaccination -other serotypes and NTHi more visible

  9. Hhvs Hi • Non-encapsulated (non-typeable) Hi: • Colonization is confined to the nasopharynx and upper airway • Cause opportunistic infections in patients with physiological defects • Common NTHi infections are localized to respiratory tract • -otitis media in children, • -lower respiratory tract infections (acute tracheobronchitis, pneumonia) in children and adults • -colonizer in COPD patients

  10. Hh: • Two cases of endocarditis (1920-30) • ABCs cases • 5 cases (out of 392 Hi cases) from 2009-2010 • 4 cases (out of 234 Hi cases) from 1999-2000 • Respiratory disease??

  11. Hhae EU185340 Hhae EU185348 66 Hhae EU185349 16S rRNA gene sequencing analysis Hhae EU185354 100 case4 Hhae EU185339 51 79 Hhae EU185350 case1 61 Hhae S0000436674 Hhae EU185316 60 case5 55 Hhae EU185345 48 case2 case3 100 Hhae EU185337 H. paraphrohaemolyticusS000436705 32 H. parainfluenzae AY365452 57 H. parainfluenzae AY365450 100 H. parainfluenzae AY362908 94 66 H. parainfluenzae EU083530 H. parasuisAB004024 Hi AY613546 62 Hi AY613743 Hi AY613451 91 Hi AY613586 99 Hi AY613724 53 Hi AY613739 Hi AY613720 87 44 Hi AY613728 Hi AY613480 63 Hi AY613535 51 Hi AY613446 Hi AY613510 Hi AY613500 39 Hi AY613591 Hi AY613468 62 Hi AY613493 0.005 Hi AY613474 Hi AY613482

  12. 1. Genetic diversity of Hh and Hi and causing mechanisms 2. Functional analysis of Hh and Hi genomes

  13. Delineation of bacterial species by genetic relatedness • DNA-DNA hybridization: degree of re-association of single-stranded DNA. Isolates that show 70% or more DNA hybridization are defined as the same species. • Others: 16S rRNA gene, MLST genes and/or infB • 16S rRNA gene sequence similarity: If the similarity is 98.5% (97% previously) or less, the corresponding DNA re-association value is always lower than 70%. • Genetic variation caused by vertical and horizontal gene transfer complicated species definition. This cutoff may not apply for closely related species.

  14. Intra-species genetic diversity • ~25% difference in gene content among N. meningitidis, Helicobacter pylori and E. coli • Genetic diversity of NTHi • Out of 242 strains analyzed by Multilocus enzyme elelctrophoresis, all 65 NTHi have a distinct electrophoretic type (ET), 177 typeable Hi belong to 29 ETs • Not clonal by MLST • Distinguish intra- and inter- species genetic diversity.

  15. Mechanisms of genetic diversity: driven by vertical and horizontal gene transfer • Vertical • Point mutation (light or chemical induced) • Inversion • Spontaneous deletion • Horizontal • Transformation (chromosomal DNA) • Transposon mutagenesis • Transduction (phage) • Conjugation (plasmid) • Transformation (plasmid)

  16. Transformation • A process of direct uptake, incorporation and expression of exogenous genetic material from its surrounding. • Plasmid, exogenous chromosomal DNA fragments, and transposon. • Natural transformation/chemical or electrical transformation. • Hi is naturally competent. Genes involved in this process have been identified. Competency genes in Hhae?

  17. Transponson mutagenesis Transposons: mobile DNA segments that can disrupt gene function by inserting in or near genes. Also referred as IS element.Pay attention to genes flanked by IS elements.

  18. Questions Accuracy of using16S rRNA gene sequence, MLST alleles and/or infBfor delineation of bacterial species Genetic diversity of Hh/Hi (intra- and inter- speices) Mechanisms causing genetic diversity Others?

  19. Genome (genes/regulatory elements) Genotype Transcription mRNA Translation Proteins (enzymes, virulent factors, surface structures, and others) Phenotype

  20. Function analysis of Hh genome: Genotypes (or phenotypes) discriminating Hi and Hh • Potential targets: Orfs unique to Hh or Hi • Gene(s) encoding beta-hemolysis • -One standard way of distinguishing between Hi and Hh is that Hh is haemolytic on horse blood agar plate.  Genes encoding this function is unknown. • -Cases of nonhaemolyticH. haemolyticus are becoming more common. Is the loss of this activity reversible? What is the regulatory mechanism of this conversion?

  21. Other bacteria undergoing beta-hemolysis • Staphylococcus aureus: beta-haemolysin gene (hlb) was composed of 993 nucleotides encoding a mature polypeptide 330 amino acids. • Streptococcus pyogenes (Group A streptococcus): SLS, cell-bound cytolycin • S. pneumoniae are alpha-hemolytic but can cause ß-hemolysis during anaerobic incubation. • GBS: β-h/c is a pore-forming membrane-associated toxin that promotes injury of a broad range of eukaryotic cell types • Haemophilusducreyi: variable hemolytic activity or alpha-hemolysis homologue of hpmA/B of P. mirabilis • Targets: secreted proteins, OMPs or lipoproteins • Suggested reading: TRENDS in Microbiology Vol.10 No.12: 575 • INFECTION AND IMMUNITY, 63(11) 1995, p. 4409–4416

  22. Proposed biomakers • iga, lgtC, fucK and hpd. similarity of these genes within species and diversity between Hh and Hi. Not extensively validated. • Novel biomakers ????

  23. Pathogenesis of NTHi Colonization (surface structures) Evasion of Host Defense

  24. Gram-negative bacterial surface structures

  25. Hi virulence factors 1. Adherence: confined to the nasopharynx and upper airway Pili (hifABCDE): bind to respiratory mucus and human oropharyngeal epithelial cells Hap (Haemophilus adhesion and penetration, hap) serine protease; promote adherence and invasion Hia/Hsf (hia/hsf): adherence; fimbrial structure; High-affinity adhesive activity and mediates interaction with a broad array of respiratory epithelial cell types

  26. HMW1/HMW2 (high-molecular-weight proteins; hmw1A, B, C. hmw2A, B, C ) To date, the hmw genes have only been detected in nontypable Himediate attachment to human epithelial cells, an essential step in the pathogenesis of disease. Outer membrane P2, P5 and others Lipopolysaccharide (LPS): adherence, invasion. and damage (endotoxin) extensive inter-strain and intra-strain heterogeneity of glycoform structure which is key to the role of the molecule in both commensal and disease-causing behavior of the bacterium.

  27. 2. Immune Evasion IgA protease: three types of IgA in Hi capsule (the cap locus): Phase variation and antigen variation 3. Entry into host cells Capsule-deficient Hi strains invade endothelial cells and remain in vacuoles over an extend period. Mechanism unknown. 4. Damage Hemolysin, LPS

  28. Others functions Iron acquisition DNA modification and restriction systems (maintain species-specificity) Oxidative stress Surface structures: Outer membrane protein, pili etc Secretion systems (transporter) contingency genes: two-component systems Carbohydrate and amino acid metabolism pathways DNA and protein biosynthesis and degradation Suggested reading: Genomic sequence of an otitis media isolates of nontypeable Haemophilus influenzae: comparative study with H. influenzae serotype d, strain KW20. Journal of Bacteriology 187 (13): 4627-4636

  29. Thanks for your attention. Questions?

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