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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 • Positive for cytochrome oxidase • Growth in culture requires exogenous hemin (oxidized ferroprotoporphyrin) (X factor) and/or nicotinamide adenine dinucleotide (NAD) (V factor) Haemophilus influenzae 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 Hh vs NTHi 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 Identification of Hi and Hh Colony morphology on selective media Kovac’s oxidase test to determine the presence of cytochrome oxidase 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) Phenotype Require X and V factor for growth Hemolysis H2S production Sugar metabolism H. influenzae NT H. haemolyticus Yes Yes - +/- 90% - 90% + Glu +, (Sur, Lac, Man)- Glu +, (Sur, Lac, Man)- Urease, Ornithine decarboxylase (ODC) +/- +/- Indole produce +/- +/- H. Influenzae Genotype by PCR or DNA blot H. haemolyticus NT iga (IgA protease gene) + -/+?? fucK (fuculous kinase gene) +/- -? hpd (protein D gene) +/- -? lgtC (LPS gene) +/- +/- A new simple typing scheme is needed! 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 Hh vs Hi Hi and Hh are 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 Hi was most commonly prior to Hib conjugate vaccine -prevalence of Hib cases has declined with advent of vaccination -other serotypes and NTHi more visible Hh vs 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 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?? Case 1 Case 2 Case 3 Case 4 Case 5 Sex female female female male male Age 47 6 63 19 60 Underlying condition(s) smoker, intravenous drug user, cirrhosis, alcoholism, Hepatitis C positive none diabetes mellitus, obesity gunshot wound Knee surgery Diagnosis bacteremia without focus bacteremia without focus peritonitis syndrome bacteremia Knee infection V factor requirement + + + + + X factor requirement + + + + + Hemolysis + + + - - H2S production + + + + + hpd PCR negative negative negative negative negative fucK PCR negative negative negative negative negative Identification by 16S rRNA gene Hhae Hhae Hhae Hhae Hhae No Hh was isolated from CSF 16S rRNA gene sequencing analysis Hhae EU185340 Hhae EU185348 66 Hhae EU185349 Hhae EU185354 100 case4 Hhae EU185339 51 79 Hhae EU185350 case1 61 Hhae S0000436674 Hhae EU185316 60 55 case5 48 Hhae EU185345 case2 100case3 Hhae EU185337 H. paraphrohaemolyticus S000436705 32 H. parainfluenzae AY365452 57 100 H. parainfluenzae AY365450 94 H. parainfluenzae AY362908 66 H. parainfluenzae EU083530 H. parasuis AB004024 Hi AY613546 62 Hi AY613743 Hi AY613451 91 Hi AY613586 99 Hi AY613724 53 Hi AY613739 87Hi AY613720 44 Hi AY613728 Hi AY613480 63 Hi AY613535 51 Hi AY613446 Hi AY613510 39 Hi AY613500 Hi AY613591 Hi AY613468 62 Hi AY613493 0.005 Hi AY613474 Hi AY613482 1. Genetic diversity of Hh and Hi and causing mechanisms 2. Functional analysis of Hh and Hi genomes Delineation of bacterial species by genetic relatedness DNA-DNA hybridization: degree of re-association of singlestranded 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. 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. 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) 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? 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. Questions Accuracy of using16S rRNA gene sequence, MLST alleles and/or infB for delineation of bacterial species Genetic diversity of Hh/Hi (intra- and inter- speices) Mechanisms causing genetic diversity Others? Genome (genes/regulatory elements) Genotype Transcription mRNA Translation Proteins (enzymes, virulent factors, surface structures, and others) Phenotype 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 nonhaemolytic H. haemolyticus are becoming more common. Is the loss of this activity reversible? What is the regulatory mechanism of this conversion? 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 Haemophilus ducreyi: 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 Proposed biomakers iga, lgtC, fucK and hpd. similarity of these genes within species and diversity between Hh and Hi. Not extensively validated. Novel biomakers ???? Pathogenesis of NTHi Colonization (surface structures) Evasion of Host Defense Gram-negative bacterial surface structures 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 HMW1/HMW2 (high-molecular-weight proteins; hmw1A, B, C. hmw2A, B, C ) To date, the hmw genes have only been detected in nontypable Hi mediate 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. 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 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