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.

http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6WG1-50K5SYH-1&_image=B6WG1-50K5SYH-1-F&_ba=&_user=856389&_coverDate=11/30/2010&_rdoc=1&_fmt=full&_orig=gateway&_cdi=6809&_pii=S0888754310001588&view=c&_isHiQual=Y&_acct=C000046148&_version=1&_urlVersion=0&_userid=856389&md5=b8c564756caf4d4fe9b2c8f83af9ec21

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?