Transcript Careers in Public Health Program Promoting and Supporting

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