INFECTION AND IMMUNITY, May 2011, p. 1971–1983
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 5
ArcA-Regulated Glycosyltransferase Lic2B Promotes Complement
Evasion and Pathogenesis of Nontypeable
Sandy M. S. Wong,1Frank St. Michael,3Andrew Cox,3Sanjay Ram,2and Brian J. Akerley1*
Department of Microbiology and Physiological Systems1and Division of Infectious Diseases and Immunology,2University of
Massachusetts Medical School, Worcester, Massachusetts 01655, and Institute for Biological Sciences,
National Research Council, Ottawa, Ontario, Canada KIA 0R63
Received 1 December 2010/Returned for modification 30 December 2010/Accepted 16 February 2011
Signaling mechanisms used by Haemophilus influenzae to adapt to conditions it encounters during stages of
infection and pathogenesis are not well understood. The ArcAB two-component signal transduction system
controls gene expression in response to respiratory conditions of growth and contributes to resistance to
bactericidal effects of serum and to bloodstream infection by H. influenzae. We show that ArcA of nontypeable
H. influenzae (NTHI) activates expression of a glycosyltransferase gene, lic2B. Structural comparison of the
lipooligosaccharide (LOS) of a lic2B mutant to that of the wild-type strain NT127 revealed that lic2B is
required for addition of a galactose residue to the LOS outer core. The lic2B gene was crucial for optimal
survival of NTHI in a mouse model of bacteremia and for evasion of serum complement. The results demon-
strate that ArcA, which controls cellular metabolism in response to environmental reduction and oxidation
(redox) conditions, also coordinately controls genes that are critical for immune evasion, providing evidence
that NTHI integrates redox signals to regulate specific countermeasures against host defense.
Haemophilus influenzae is a Gram-negative bacterium that
colonizes the human nasopharyngeal mucosa and can dissem-
inate to other sites to cause otitis media, upper and lower
respiratory tract infections, septicemia, and meningitis (37, 50).
It frequently infects the lungs of individuals with chronic ob-
structive pulmonary disease (51, 52, 65) and cystic fibrosis (20,
49). The introduction in 1990 of an effective vaccine against the
capsular polysaccharide of encapsulated H. influenzae type b
(Hib) strains has decreased the incidence of systemic infections
caused by Hib strains in developed countries (9). However, the
vaccine is not effective against nonencapsulated, nontypeable
H. influenzae (NTHI). NTHI predominantly causes respiratory
tract infections and otitis media but occasionally can enter the
bloodstream to cause meningitis (11, 15, 54, 55). Prior to in-
troduction of the Hib vaccine, NTHI was not a major cause of
invasive disease; however, in the post-Hib vaccine era, the
incidence of invasive infections due to NTHI has increased and
is shifting from infants to older populations (14, 68).
The factors contributing to invasive disease, likely involving
host susceptibility and strain-specific virulence genes, are not
well understood. A correlation was observed between disease
severity during invasive NTHI infections (bacteremia or men-
ingitis) and the degree of resistance of the corresponding
NTHI isolate to bactericidal effects of human serum in vitro,
suggesting that increased resistance to complement enhances
virulence (23). The complement system plays an important role
in adaptive and innate immune defenses against H. influenzae
infection in both humans and animal models (16, 17, 59, 72,
74). Three major pathways, i.e., classical, lectin, and alterna-
tive, that differ in their mode of activation on the pathogen
surface (60, 72) can initiate complement deposition. Each
pathway involves a cascade of proteolytic cleavage steps that
activate subsequent factors, leading to antimicrobial activities
that include target cell lysis, inflammation, opsonization-pro-
moting phagocytosis, and activation of the bactericidal mech-
anisms of macrophages and neutrophils.
The lipopolysaccharide (LPS) glycolipid of the outer leaflet
of the Gram-negative bacterial outer membrane mediates eva-
sion of the complement system and is essential in animal mod-
els of invasive infection by H. influenzae (7, 16, 29, 41). In
H. influenzae and in many other human respiratory tract patho-
gens, the LPS is termed lipooligosaccharide (LOS) because it
lacks the repetitive polysaccharide O-antigen side chain pres-
ent in the LPS of other Gram-negative bacteria (50). The LOS
structure varies between strains, yet several features are con-
served. H. influenzae LOS consists of lipid A, an inner core
usually composed of a single 3-deoxy-D-manno-octulosonic
acid linked to three heptose residues, and an outer core usually
containing a short heteropolymer of glucose and galactose
residues in different configurations extending from the hepto-
syl residues of the inner core. The outer core may additionally
be modified with sialic acid, N-acetylgalactosamine, and phos-
phorylcholine (31, 62).
During pathogenesis, bacteria sense and respond to environ-
mental signals to appropriately express critical virulence fac-
tors or to repress those that would otherwise detract from
efficient infection, such as structures recognized by host im-
mune pathways. H. influenzae has been shown to modify its
LOS in response to environmental aeration conditions by in-
creasing levels of phosphorylcholine displayed on the LOS
outer core as oxygen levels decrease (75), a response that may
* Corresponding author. Mailing address: Department of Molec-
ular Genetics and Microbiology, University of Massachusetts Med-
ical School, 55 Lake Ave. N., S6-242, Worcester, MA 01655. Phone:
(508) 856-1442. Fax:(508) 856-1422.
?Published ahead of print on 28 February 2011.
allow NTHI to differentially express LOS structures for eva-
sion of immune effectors present in environments in the host
such as airway mucosal surfaces versus invasion into deeper
tissues or in the bloodstream. Mechanisms by which H. influ-
enzae senses and responds to such reduction/oxidation (redox)
signals to regulate LOS synthesis have not been identified;
however, H. influenzae possesses a redox-responsive regulatory
system, the ArcAB two-component signaling system (TCS),
that is biochemically and functionally similar to that of Esch-
erichia coli (19, 44). Under low-oxygen conditions, ArcB senses
the redox status of the quinone pool and autophosphorylates,
leading to activation of ArcA by phosphoryl transfer (4, 18,
43). Phosphorylated ArcA transcriptionally activates or represses
diverse target genes, including genes of the tricarboxylic acid
cycle and genes involved in other aspects of respiratory or
fermentative metabolism (12, 39, 40, 77). Under high-oxygen
conditions, ArcAB activity is greatly decreased. In H. influen-
zae, ArcA has been demonstrated to be important for serum
resistance and pathogenesis in mouse models of bacteremia
(12, 77); however, ArcA-regulated virulence genes have not
been identified, and the mechanism of ArcA-mediated serum
resistance is unknown.
In the current study, we investigated mechanisms by which
the H. influenzae ArcAB two-component signaling system influ-
ences NTHI pathogenesis. Strain-specific differences in ArcA-
dependent serum resistance led us to investigate whether ArcA-
regulated LOS genes unique to invasive strains are involved in
this virulence phenotype. We found that ArcAB controls ex-
pression of genes involved in the production of the outer core
of the LOS, lic2B and lic2C, in NTHI strains. The LOS struc-
ture specified by Lic2B and its role in inhibition of complement
interactions were characterized to provide insight into the mo-
lecular mechanism of NTHI pathogenesis. Together the results
indicate that the redox-responsive ArcA regulator controls
transcription of LOS glycosyltransferase genes that are impor-
tant for pathogenesis and evasion of complement, suggesting
that H. influenzae utilizes redox signaling to appropriately
modify its immune evasion strategies in different environments
encountered during infection.
MATERIALS AND METHODS
Media and Haemophilus influenzae growth conditions. The nontypeable Hae-
mophilus influenzae (NTHI) clinical isolates NT127 and PittGG were grown at
35°C ? 1.5°C in brain heart infusion supplemented with 10 ?g/ml NAD and 10
?g/ml hemin (sBHI) on agar plates or in sBHI broth cultures. NT127 was
isolated from the blood of a 6-month-old patient with meningitis (provided by
Robert N. Husson) (25), and its genome has been sequenced (GenBank acces-
sion no. ACSL00000000.1). PittGG (provided by Garth D. Ehrlich), which also
has been sequenced (GenBank accession no. CP000672.1), was isolated from the
external ear discharge of a child with otorrhea (7, 66). DNA was transformed
into naturally competent H. influenzae prepared as previously described (3).
Kanamycin (Km), gentamicin (Gm), and tetracycline (Tet) were added to sBHI
at 20 ?g/ml, 10 ?g/ml, and 8 ?g/ml, respectively.
Plasmid and H. influenzae strain construction. Standard molecular biology
methods were used for plasmid construction (2). All primer sequences are listed
in Table 1. Strains and plasmids used in this study are listed in Table 2. An arcA
mutation in NT127 was created by amplification of a 3.56-kb PCR product using
primers ArcA2466 and ArcA5582 from H. influenzae Rd strain RAA6, which
contains a nonpolar, in-frame deletion of the arcA protein-coding sequences
(19), and transformation of NT127 with the resulting product. Kmrtransfor-
mants were selected on sBHI agar containing Km to create the arcA deletion
strain NTAA. Strains NT127V and NTAAV, containing empty vector sequences
at the partial xyl locus, were generated as follows. A 1.4-kb PCR product was
amplified from NT127 with primers JbaspC2-Pci and xylAorfout, and an 6.8-kb
PCR product was amplified from pXT10 (76) with primers tetR-in1 and xylB-
3ORF3. These two products were used as templates in a PCR stitching reaction
to generate a 8.2-kb PCR product for transformation into NT127 and NTAA.
Tetrtransformants undergoing homologous recombination between the 8.2-kb
product at aspC2 and the xyl locus were selected on sBHI agar containing Tet. An
arcA complementing strain containing a wild-type copy of arcA in the xylA locus
was created as follows. The 1.4-kb PCR product described above and a 7.73-kb
PCR product amplified from pXTAA (19) with primers AAtetR-in1 and xylB-
3ORF3 were used as templates in a PCR stitching reaction to generate a 9.1-kb
PCR product used for transformation into NTAA. Tetrtransformants were
selected on sBHI agar containing Tet to create the arcA complementing strain
NTAAC. Nonpolar, in-frame deletion mutations of lic2B in NT127 were created
by replacement of the protein-coding sequences with the aacC1 gentamicin
resistance cassette to create NTlic2B by PCR stitching as follows. A 1,023-bp
PCR product containing the 5? flanking region of lic2B was amplified from
NT127 with primers 1kb5?JBlic2B and JBlic2B-5?out. A 1,030-bp PCR product
containing the 3? flanking region of lic2B was amplified from NT127 with primers
JBlic2B-3?out and 1kb3?JBlic2B. A 536-bp fragment containing the aacC1 gen-
tamicin resistance gene was amplified with primers aacC15? and aacC13? from
pBSL182 (1). The 1023-bp, 1030-bp, and 536-bp products were stitched in a PCR
with primers 1kb5?JBlic2B and 1kb3?JBlic2B. The resultant 2,559-bp product was
introduced into three independent cultures of NT127, and Gmrtransformants
were selected on sBHI agar containing Gm to create independent isolates of
strain NTlic2B containing a precise replacement of the lic2B coding sequence
with those of aacC1. Similarly, a lic2B deletion mutation in PittGG was created
by transforming this strain with the same 2,559-bp PCR product (see above) and
selecting for Gmrtransformants on sBHI agar containing Gm to create strain
A nonpolar, in-frame deletion of lic2C in NT127 was created by replacement
of the protein-coding sequences with the aacC1 gentamicin resistance cassette to
create NTlic2C by PCR stitching as follows. A 1,023-bp PCR product containing
the 5? flanking region of lic2C was amplified from NT127 with primers
1kb5?JBlic2C and JBlic2C-5?out. A 1,036-bp PCR product containing the 3?
flanking region of lic2C was amplified from NT127 with primers JBlic2C-3?out
and 1kb3?JBlic2C. The 1,023-bp, 1,036-bp, and 536-bp products containing the
aacC1 gentamicin resistance cassette (see above) were combined by sequence
overlap extension PCR with primers 1kb5?JBlic2C and 1kb3?JBlic2C. The resul-
tant 2,565-bp product was introduced into NT127, and Gmrtransformants were
selected on sBHI agar containing Gm to create strain NTlic2C.
A nonpolar, in-frame deletion of lex2A in NT127 was created by replacement
of the protein-coding sequences with a kanamycin resistance gene to create
NTlex2A by PCR stitching as follows. A 1,019-bp PCR product containing the 5?
flanking region of lex2A was amplified from NT127 with primers 1kb5?JBlex2A
and JBlex2A-5?out. A 1,025-bp PCR product containing the 3? flanking region of
lex2A was amplified from NT127 with primers JBlex2A-3?out and 1kb3?JBlex2A.
An 818-bp fragment containing the kanamycin resistance gene aphI from Tn903
(76) was amplified with primers kan5?ATG and kan3??TAA. The 1,019-bp,
1,025-bp, and 818-bp products were combined in a PCR with primers
1kb5?JBlex2A and 1kb3?JBlex2A. The resultant 2,832-bp product was introduced
into NT127, and Kmrtransformants were selected on sBHI agar containing Km
to create strain NTlex2A.
The lic2B-complemented strain NTlic2Bcomp was generated by using our
exchange vector pXT10 (76) containing the wild-type copy of lic2B for homol-
ogous recombination into the xyl locus. Briefly, a 1,081-bp product containing the
lic2B gene and its promoter region was amplified from NT127 with primers
lic2Bcomp5 and lic2Bcomp3. The 1,081-bp PCR product was digested with SapI
and cloned into the SapI sites of pXT10 to create plic2Bcomp. Because strain
NT127 lacks xylFGH, which are needed for recombination with pXT10, we
generated a Kmr-marked derivative of NT127 that contains the complete xyl
locus, as described previously (25), for subsequent transformation with pXT10
and its derivatives. The Kmr-marked NT127 derivative was transformed with the
2,559-bp PCR product containing the lic2B mutation (see above) to create a
lic2B mutant in this background. This resultant strain was then transformed with
pXT10 and the complementing plasmid, plic2Bcomp, to generate NTlic2BV and
NTlic2Bcomp, respectively. The Kmr-marked NT127 derivative was also trans-
formed with pXT10 to create a parent strain, NTV, carrying the empty cloning
vector (25). All strain constructions were verified by PCR amplification across
the inserted recombinant region with primers specific for flanking sequences.
Genes introduced for complementation of mutations were verified by DNA
Serum bactericidal assays. Serum bactericidal testing was performed as de-
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