Post on 24-Feb-2016
description
Exploring Haemophilus haemolyticus by
functional genomic analyses
Xin Wang, Ph. D
Meningitis Laboratory, CDC
MVPDB/DBD/NCIRDWHO 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 NTHiHh shares high similarity with Hi:
Colony and cell morphologyCarbohydrate metabolismBiochemical featuresGenetic backgroundDoesn’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 HhColony morphology on selective media Kovac’s oxidase test to determine the presence of cytochrome oxidase Porphyrin test : exclude hemin-independent Haemophilus strainsHemo 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 H. influenzae NT
H. haemolyticus
Require X and V factor for growth
Yes Yes
Hemolysis - +/-H2S production 90% - 90% +Sugar metabolism
Glu +, (Sur, Lac, Man)-
Glu +, (Sur, Lac, Man)-
Urease, Ornithine decarboxylase (ODC)
+/- +/-
Indole produce +/- +/-
Genotype by PCR or DNA blot
H. Influenzae
NT
H. haemolyticus
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 assaysPCR: hoursgene sequencing: days
Genome-based assays: days or weeks
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 defectsCommon 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 vs Hi
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??
Hhae EU185340Hhae EU185348Hhae EU185349Hhae EU185354case4Hhae EU185339
Hhae EU185350
66100
case1Hhae S0000436674Hhae EU185316
case5Hhae EU185345
case2case3
Hhae EU185337H. paraphrohaemolyticus S000436705
H. parainfluenzae AY365452H. parainfluenzae AY365450
H. parainfluenzae AY362908H. parainfluenzae EU083530H. parasuis AB004024
Hi AY613546Hi AY613743
Hi AY613451Hi AY613586Hi AY613724Hi AY613739
Hi AY613720Hi AY613728Hi AY613480Hi AY613535Hi AY613446Hi AY613510Hi AY613500Hi AY613591Hi AY613468Hi AY613493Hi AY613474Hi AY613482
5179
61
60 5548100
3257
1009466
6291
5399
874463
51
39
620.005
16S rRNA gene sequencing analysis
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 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.
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)
TransformationA 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 mutagenesisTransposons: 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.
QuestionsAccuracy 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
mRNATranslation
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-hemolysisStaphylococcus 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 lipoproteinsSuggested reading: TRENDS in Microbiology Vol.10 No.12: 575
INFECTION AND IMMUNITY, 63(11) 1995, p. 4409–4416
Proposed biomakersiga, 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 factors1. 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 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.
2. Immune EvasionIgA protease: three types of IgA in Hicapsule (the cap locus):Phase variation and antigen variation
3. Entry into host cellsCapsule-deficient Hi strains invade endothelial cells and remain in vacuoles over an extend period. Mechanism unknown.
4. DamageHemolysin,LPS
Others functions Iron acquisitionDNA modification and restriction systems
(maintain species-specificity)Oxidative stressSurface structures: Outer membrane
protein, pili etcSecretion systems (transporter)contingency genes: two-component
systemsCarbohydrate and amino acid metabolism
pathwaysDNA 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
Thanks for your attention.Questions?