Molecular pathogenesis of Klebsiella pneumoniae

Abstract

ABSTRACT Typical Klebsiella pneumoniae is an opportunistic pathogen, which mostly affects those with weakened immune systems and tends to cause nosocomial infections. A subset of hypervirulent K. pneumoniae serotypes with elevated production of capsule polysaccharide can affect previously healthy persons and cause life-threatening community-acquired infections, such as pyogenic liver abscess, meningitis, necrotizing fasciitis, endophthalmitis and severe pneumonia. K. pneumoniae utilizes a variety of virulence factors, especially capsule polysaccharide, lipopolysaccharide, fimbriae, outer membrane proteins and determinants for iron acquisition and nitrogen source utilization, for survival and immune evasion during infection. This article aims to present the state-of-the-art understanding of the molecular pathogenesis of K. pneumoniae.

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REVIEW
Molecular pathogenesis of Klebsiella
pneumoniae
Bei Li‡,1, Yuling Zhao‡,2, Changting Liu3, Zhenhong Chen*,3 &DongshengZhou*,4
1Department o f Dermatology, Taihe Hospital, Hubei University of Med icine, Shiyan, Hubei, China
2The 89th hosp ital of People’s Liberation Army, Weifang, Shandong, China
3Nanlou Respir atory Diseases Depar tment, Chinese People’s Liber ation Army General Hospital, Beijing, China
4State Key Laborator y of Pathogen & Biosecurity, Beijing Instit ute of Microbiology & Epidemiology, Beijing, China
*Authors for correspondence: dongshengzhou1977@gmail.com; zhenhong_chen@hotmail.com
‡Authors contributed equally
ABSTRACT Typ ica l Klebsiella pneumoniae is an opportunistic pathogen, which mostly
aects those with weakened immune systems and tends to cause nosocomial infections.
A subset of hypervirulent K. pneumoniae serotypes with elevated production of capsule
polysaccharide can aect previously healthy persons and cause life-threatening community-
acquired infections, such as pyogenic liver abscess, meningitis, necrotizing fasciitis,
endophthalmitis and severe pneumonia. K.pneumoniae utilizes a variety of virulence factors,
especially capsule polysaccharide, lipopolysaccharide, mbriae, outer membrane proteins
and determinants for iron acquisition and nitrogen source utilization, for survival and immune
evasion during infection. This article aims to present the state-of-the-art understanding of
the molecular pathogenesis of K.pneumoniae.
KEYWORDS
• Klebsiella pneumoniae
• pathogenesis • virulence
determinants
Klebsiella pneumoniae, a member of the family Enterobacteriaceae, is a rod-shaped, Gram-negative,
lactose-fermenting bacillus with a prominent capsule. Typical K. pneumoniae is an opportunistic
pathogen that is widely found in the mouth, skin and intestines, as well as in hospital settings and
medical devices. Opportunistic K. pneumoniae mostly affects those with compromised immune
systems or who are weakened by other infections. Colonization of the GI tract by opportunistic
K. pneumoniae generally occurs prior to the development of nosocomial infections, and K. pneu-
moniae colonization can be further found in the urinary tract, respiratory tract and blood [1] .
K. pneumoniae biofilms that form on medical devices (e.g., catheters and endotracheal tubes) pro-
vide a significant source of infection in catheterized patients [2] . Nosocomial infections caused by
K. pneumoniae tend to be chronic due to the two following major reasons: K. pneumoniae biofilms
formed in vivo protect the pathogen from attacks of the host immune responses and antibiotics [3] ;
and nosocomial isolates of K. pneumoniae often display multidrug-resistance phenotypes that are
commonly caused by the presence of extended-spectrum β-lactamases or carbapenemases, making
it difficult to choose appropriate antibiotics for treatment [4,5] .
At least 78 capsular (K antigen) serotypes have been recognized for K. pneumoniae [6 –8] . A few
serotypes (including predominantly K1 and K2) have a unique hypermucoviscous (hypervirulent)
phenotype due to increased production of capsule polysaccharide (CPS), which is recognized as
the most important virulence factor of K. pneumoniae (see below) and is defined by the appear-
ance of hypermucoviscous colonies grown on agar plates [9 ,10]. A string test indicates the presence
of hypermucoviscosity when a inoculation loop is able to generate a viscous string larger than
5 mm in length by stretching bacterial colonies on agar plate [1 0] . The degree of mucoidy appears
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to positively correlate with the successful estab-
lishment of invasive infections [11] . Nevertheless,
whether there is hypersecretion of capsule in the
hypervirulent strains remains questionable, as
the increased amount of polysaccharide observed
might be some exopolysaccharides rather than
‘truly organized’ CPS sugars.
Hypervirulent K. pneumoniae is highly
invasive and can affect previously healthy per-
sons, causing life-threatening and often com-
munity-acquired infections, such as pyogenic
liver abscess, meningitis, necrotizing fasciitis,
endophthalmitis and severe pneumonia [9,10].
In particular, serotype K1/K2-caused pyogenic
liver abscess, often complicated by metastatic
infections, has emerged worldwide in the past
two decades [12 ] . The ability to metastatically
spread from one organ to other organs is charac-
teristic of hypermucoviscous K. pneumoniae and
is uncommon for enteric Gram-negative bacilli
in the presence of host immune defense.
To the best of our knowledge, there are no
full reviews on the molecular pathogenesis of
K. pneumoniae. This article presents the state-
of-the-art understanding of the virulence deter-
minants of K. pneumoniae, with a particular
emphasis on hypervirulent K. pneumoniae.
Capsule polysaccharide
●●CPS gene loci
K. pneumoniae CPS is an acidic polysaccharide
generally composed of repeating units of three
to six sugars. K. pneumoniae CPS is synthesized
by the Wzy-dependent polymerization pathway,
which is well characterized for Escherichia coli
group I CPS [13 ,14], and the cps gene clusters
commonly consist of genes for sugar nucleo-
tide synthesis, capsule repeat-unit synthesis and
capsular repeat-unit assembly and export [15 ,16] .
The full length of the K. pneumoniae cps gene
clusters ranges from 21 to 30 kb, harboring
16–25 genes (see [15 ,16] for the gene structure
of different cps gene clusters). The 5´ terminal
regions of all known K. pneumoniae cps gene
clusters contain six conserved genes (in the fol-
lowing order: galF, orf2, wzi, wza, wzb and wzc)
and the 3´ end regions contain a conserved gene
gnd and are mostly terminated at the ugd gene,
while the central regions, which encode the
proteins for the polymerization and assembly of
CPS subunits, are highly divergent [15,16]. Both
wzy and wzi genes are present in all K types,
but each of them has a high level of sequence
variability among distinct K types. Accordingly,
wzy-targeting PCR assays have been established
to identify K1, K2, K3, K5, K20, K54 and K57
[17,18] , and moreover, wzi sequencing is able to
define the K types of most clinical K. pneumo-
niae isolates [19 ] . In addition, the capsular typ-
ing method based on wzc sequencing has been
used for the detection of novel capsular types
of K. pneumoniae [7] .
●●CPS synthesis & magA
CPS synthesis initiates with the assembly of indi-
vidual sugar-repeat units, which are catalyzed
by different glycosyltransferases in a sequential
manner [1 3, 14] . The resulting nascent repeats
are transferred across the inner membrane by a
flippase Wzx and undergo polymerization by a
Wzy polymerase in the periplasmic space [1 3 ,14] .
Further polymerization control and export of
mature CPS to the bacterial cell surface occur
under the combined action of Wza (an inner
membrane tyrosine autokinase), Wzb (an pro-
tein-tyrosine phosphatase) and Wzc (an inte-
gral outer membrane lipoprotein) [13 ,14] . magA
(wzyKpK1) encodes a capsular K1-specific Wzy
polymerase [20 ], but plays no role in lipopoly-
saccharide (LPS) synthesis [2 1] and acts as an
important virulence determinant in experi-
mental K. pneumoniae K1-induced metastatic
infections [22] .
●●rmpA/rmpA2
Plasmid-borne rmpA and its isoform rmpA2
encode the transcriptional activators of cps gene
transcription, CPS synthesis and hypermucovis-
ity in K. pneumoniae K1/K2 [23 –25] . In the K2
strain CG43, both rmpA and rmpA2 are acti-
vators of CPS biosynthesis and are important
for virulence in mice [23 ,24] . In the K1 strain
NTUH-K2044, besides plasmid-borne rmpA
and rmpA2, there is still an rmpA paralog on
the chromosome, and only plasmid-borne rmpA
enhances the cps gene transcription [25] .
●●Resistance to phagocytosis
The presence of a thick capsule at cell surface
protects K. pneumoniae from opsonization and
phagocytosis by macrophages [26 ], neutrophils
[27] , epithelial cells [28] and dendritic cells (DCs)
[29] by blocking binding and internalization
(Figure 1). Hypervirulent K. pneumoniae K1
shows significantly lower levels of interaction
with macrophages compared with nonhyper-
virulent strains [3 0] , and moreover, the unusual
feature of extensive pyruvation of glucuronic

1073
Figure 1. Klebsiella pneumoniae-mediated immune evasion. Klebsiella pneumoniae employs various surface structures (e.g., CPS, LPS,
OmpA, OmpK36 and AcrAB) to evade host immune defenses, enabling the bacteria to resist complement-mediated killing, the action
of host-derived antimicrobial peptides and the phagocytosis of epithelial cells, macrophages, neutrophils and DCs. This enables them
to escape from neutrophil-mediated intracellular killing of engulfed bacteria, to impair the production of the proinammatory cytokine
IL-8 and antimicrobial peptides hBD2 and hBD3 by airway epithelial cells and to inhibit the maturation of DCs.
CPS: Capsule polysaccharide; DC: Dendritic cell; LPS: Lipopolysaccharide; NK: Natural killer.
Molecular pathogenesis of Klebsiella pneumoniae REviEW
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acid and acetylation of C2-OH (or C3-OH)
of fucose in K1 CPS may help K. pneumo-
niae to avoid phagocytosis [31,3 2]. In addition,
hypervirulent K1 K. pneumoniae, after being
phagocytosed by neutrophils, can steadily
escape from neutrophil-mediated intracel-
lular killing and transport the K1 bacteria to
distant sites, such as the liver, causing abscess
formation (Figure 1) [30].
●●Suppression of early inammatory
response
Airway epithelial cells produce Toll-like recep-
tors (TLRs) in order to recognize conserved
molecules expressed by pathogens, which in
turn activate signaling pathways for produc-
ing antimicrobial molecules, such as human
β-defensins (hBDs; see below) and costimula-
tory molecules and for releasing cytokines and
chemokines (Figure 1). In sharp contrast to the
infection of wild-type capsulated K. pneumo-
niae strains that characteristically lacking the
above early inflammatory response, avirulent
CPS mutants activate a potent inflammatory
program [33 ,34] . Mechanistically, the anti-
inflammatory effect of CPS is characterized by
inhibition of IL-8 expression through inhib-
iting TLR2 and TLR4 signaling [33, 34] and
NOD1-dependent pathways [35] .
●●Resistance to antimicrobial peptides
CPS at the K. pneumoniae cell surface acts as
a protective shield against the access of host-
derived antimicrobial peptides, and free CPS
released from bacterial cells can trap antimi-
crobial polypeptides in order to reduce the
amount of antimicrobial polypeptides reaching
the bacterial surface (Figure 1) [36 ,37] . Moreover,
sublethal concentrations of antimicrobial pep-
tides in the airway induce cps gene expression,
which in turn protects bacteria against the
action of antimicrobial polypeptides [37] . hBDs
produced by airway epithelial cells are potent
antimicrobial peptides; hBD1 is constitutively
Action of host-derived
antimicrobial peptides
Production of hBD2
and hBD3 by airway
epithelial cells
Production of IL-8
by airway epithelial cells
Neutrophil chemotaxis
Phagocytosis of
epithelial cells Phagocytosis of
macrophages
Phagocytosis and
maturation of DC cells
TNF-αIL-12 and TNF-α
Phagocytes The adaptive immunity
Inflammatory cytokines
NK cell migration
Distant metastases
Intracellular killing
CPS, LPS
OmpA, OmpK36
CPS CPS Phagocytosis
of neutrophils
Complement-mediated
killing
CPS, LPS,
OmpA
CPS, LPS,
OmpA, AcrAB
Klebsiella pneumoniae

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expressed, whereas the expression of hBD2 and
hBD3 is inducible by pathogens and proinflam-
matory cytokines [3 8,39]. K. pneumoniae-bound
CPS decreases the express of hBD2 and hBD3
through preventing TLR-dependent responses
and stimulating the expression of CYLD and
MKP-1, which act as the negative regulators of
hBD expression (Figure1) [40].
●●Inhibition of DC maturation
K. pneumoniae CPS can impair DC maturation
(Figure 1) and thereby reduce the DC-mediated
production of pro-Th1 cytokines, such as IL-12
and TNF-α, which will lead to the destructive
function of immature DCs during K. pneu-
moniae antigen presentation (thereby impair-
ing T-cell activation), and moreover result in
reduced DC-mediated natural killer cell migra-
tion [29]. Taken together, inhibition of DC
maturation by K. pneumoniae CPS allows the
bacteria to avoid the host defenses and thus to
multiply in vivo more easily.
Lipopolysaccharide
LPS comprises three parts: the highly con-
served and hydrophobic lipid A anchored in the
outer membrane; the highly variable O-antigen
as the outermost component of LPS; and the
core polysaccharide connecting lipid A and
O-antigen.
●●O-antigen
At least nine O-antigen groups (O1, O2, O2ac,
O3, O4, O5, O7, O8 and O12) have been rec-
ognized in K. pneumoniae [4 1] . Biosynthesis
of O-antigen is executed by the enzymes
encoded by a six-gene wb cluster composed of
wzm, wzt, wbbM, glf, wbbN and wbbO with
respect to their transcriptional direction. The
wb cluster has a conserved gene organization,
but shows high genetic variation in correspond-
ing coding sequences, accounting for the high
chemical variability in different O-antigen
groups [4 2]. O1 is the most common serotype
among clinical K. pneumoniae isolates [4 1] , and
it is also more prevalent in hypermucoviscous
(invasive) strains than in nontissue-invasive
strains [42]. K. pneumoniae O-antigen pre-
vents access of complement components to
activators (e.g., porins and rough LPS) and
thus contributes to bacterial resistance against
complement-mediated killing (Figure 1) [43] ,
and there is a higher frequency of serum resist-
ance among O1-serotype isolates than among
non-O1-serotype isolates [42] . Interestingly, in
K. pneumoniae O-antigen-deficient strains, CPS
protects the microorganism against complement
killing (Figure 1) [44 ,45] . As shown in a murine
model of septicemia generated by intraperito-
neal injection, as well as in a murine model of
liver abscess by intragastric injection, the O1
antigen of hypermucoviscous K. pneumoniae
serotype O1:K1 plays a strong role in virulence
by conveying resistance to serum killing and
by promoting bacterial dissemination to and
colonization of internal organs after the onset of
bacteremia [42] . In a murine model of pneumo-
nia caused by hypermucoviscous K. pneumoniae
serotype O1:K2, both CPS and LPS O-antigens
are essential to blood passage of the bacteria
and the development of sepsis, but only CPS
is involved in the development of K. pneumo-
niae-induced pulmonary infections, because
CPS (but not LPS O-antigen) modulates the
deposition of C3 and protects the pathogen
against human alveolar macrophage-mediated
phagocytosis [26] . Nevertheless, O-antigen con-
tributes to lethality by increasing the propensity
for bacteremia and not by significantly chang-
ing the early course of intrapulmonary infection
[46] . The O-antigen of K. pneumoniae serotype
O5:K57 contributes to in vitro adhesion to
uroepithelial cells, and also colonization of uri-
nary tracts of rats infected by the transurethral
route [43]. As a factor regulating innate immune
responses in the lung, surfactant protein D plays
an important role in the effective inhibition of
the adhesion of K. pneumoniae to lung epithe-
lial cells through specific interaction with the
mannose-rich repeating units of the O-antigen
of this pathogen [47].
●●Core polysaccharide
Only two types (type 1 and type 2) of core
polysaccharide have been characterized for
K. pneumoniae, which were synthesized by
the products of two different 13-gene wa gene
clusters [48 ,4 9]. The type 2 wa gene cluster is
composed of hldD, waaF, waaC, wabK, waaL,
wabM, waaQ, wabG, wabH, orf10, waaA, waaE
and coaD [48] . The two wa gene clusters differ
by only two genes: type 1 has wabI and wabJ,
which encode 3-deoxy-d-manno-octulosonic
acid (Kdo) transferase and heptosyltransferase,
which are responsible for the incorporation
of the last two outer core residues Kdo and
l,d-HepI, respectively; type 2 presents the
two corresponding genes wabK and wabM,

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which are involved in the transfer of the last
two outer core Glc residues [4 8]. Accordi ngly,
the major structural difference between these
two core types occurs at the GlcN substituent
in the outer core-proximal disaccharide GlcN-
(1,4)-GalA: the GlcpN residue of the type 2
core is substituted at the O-4 position by the
disaccharide β-Glcp(1– 6)-α-Glcp(1), while
that of type 1 is replaced at the O-6 position
by a Kdo residue or by α-Hep (1– 4)- α-Kdo(2)
[48,49] . The single-gene mutants of K. pneumo-
niae LPS core synthease genes, such as waaC,
waaF, wabG and wabG, are greatly attenuated
when tested in different animal models [ 48 –5 1] ,
and show drastically reduced colonization abili-
ties in experimental urinary tract infections in
rats [50] . Hypermucoviscous K. pneumoniae
syntheses type 2 core polysaccharide [52] , and
the replacement of the type 1 core in a type 2
strain presents lower virulence than the wide-
type strain of core type 2 in a murine model
infected via the intraperitoneal route [48].
●●Lipid A
Nascent core lipid A is synthesized in the
cytoplasm by a set of conserved constitutive
enzymes, transported by the ABC transporter
MsbA and finally anchored in the outer mem-
brane. Occurring during lipid A transportation,
covalent modifications of lipid A are catalyzed
by various modification enzymes upon environ-
mental stimuli, which is involved in modulating
the virulence of a number of Enterobacteriaceae
pathogens [53] . K. pneumoniae lipid A modifi-
cation contributes to resistance to host innate
defenses, especially including resistance to anti-
bacterial peptides (Figure 1), and the mutation
of modification of the genes for this enzyme
leads to the attenuation of K. pneumoniae viru-
lence when tested in different animal models
[54 ,55] . Lipid A and core polysaccharide, but
not O-antigen are required for resistance to
phagocytosis by mouse alveolar macrophages
(Figure1) [45] , which plays an important role in
host defense against K. pneumoniae [5 6] .
Fimbriae
At least four types of fimbriae, namely type 1
fimbriae [57] , type 3 fimbriae [5 7] , Kpc fimbriae
[58] and KPF-28 adhesin [5 9] , have been char-
acterized experimentally for K. pneumoniae.
The former three, as opposed to KPF-28, are
synthesized by hypermucoviscous O1:K1 strain
NTUH-K2044 with determined complete
genome sequences [52], and NTUH-K2044 still
harbors six uncharacterized fimbrial gene loci:
kpa, kpb, kpd, kpe, kpf and kpe [5 8] .
●●Type 1 mbriae
K. pneumoniae chromosomes contain a con-
served fimbria-encoding region that is com-
posed of the regulatory gene locus mrkHIJ, the
type 3 fimbriae gene cluster mrkABCDF, a four-
gene interspacing region and the type 1 fim-
briae gene locus fimBEAICDFGHK [5 7] . Ty p e
1 fimbriae are thin, rigid, adhesive, thread-like
surface appendages on the outer membrane,
and the appendages are primarily composed
of repeating FimA subunits with an adhesin
molecule FimH at the tip [60] . Compared with
E. coli, the K. pneumoniae fim gene cluster has an
unique gene fimK located behind fimH. FimK
act as transcriptional regulator with a putative
DNA-binding region at its N-terminus and it
can specifically bind to a vegetative promoter
upstream of fimA in order to stimulate fimA
transcription [61] . A 314-bp region flanked by
inverted repeat sequences (fim switch), named
fimS, is located upstream of fimA, and moreo-
ver, fimS mediates the phase-variable expression
of K. pneumoniae type 1 fimbriae [62], which is
very similar to the expression of E. coli type
1 fimbriae [63] . K. pneumoniae type 1 fimbriae
extend beyond the capsule and mediate bacte-
rial adhesion to mannose-containing structures
on host cells or on extracellular matrices via the
adhesin FimH [64] . K. pneumoniae type 1 fim-
briae are essential for the initial establishment
of urinary tract infection, but have no effect
on the ability of K. pneumoniae to colonize the
intestine or to infect the lung [62] .
●●Type 3 mbriae
K. pneumoniae type 3 fimbriae are characterized
as 2–4-nm wide and 0.5–2-μm long append-
ages. mrkA encodes the fimbrial subunit, which
is polymerized to form the helical fimbrial shaft
[65] . The adhesive subunit, with the ability to
bind to collagen molecules, is encoded by mrkD
and located at the tip of the fimbriae [65] . mrkB,
mrkC and mrkF encode the chaperone, usher
and scaffolding proteins, respectively, which are
responsible for fimbrial assembly/stabilization
[65] . Type 3 fimbriae mediate in vitro adhesion
to epithelial cells and kidney and lung tissues,
most likely in a mannose-resistant manner [6 6] .
Type 3 fimbriae act as a major contributor to
K. pneumoniae biofilm formation, but play no

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role in intestine and pulmonary infections [5 7] .
K. pneumoniae-induced urinary tract infec-
tions are frequently associated with the forma-
tion of K. pneumoniae biofilms on indwelling
urinary catheters. Both type 1 and type 3 fim-
briae, functioning in a compensating manner,
enhance K. pneumoniae biofilm formation on
urinary catheters [67]. In addition to type 1
fimbriae, type 3 fimbriae act as another impor-
tant colonization factor for K. pneumoniae bio-
film-associated urinary infections due to the
indwelling of urinary catheters [6 8].
●●Kpc mbriae
The Kpc fimbriae are synthesized and assembled
by the products of the kpcABCD operon, which
is highly associated with hypermucoviscous
K. pneumoniae [58] . A site-specific recombinase-
encoding gene kpcI together with a promoter
element kpcS (a 302-bp intergenic DNA region
flanked by 11-bp inverted repeats) are located
upstream of kpcA [58] . KpcA is the major subunit
component of Kpc fimbriae, and KpcS in com-
bination with KpcI mediates the phase-variable
regulation of Kpc fimbriation [5 8] . Heterologous
expression of kpcABCD in a fimbriate E. coli
makes the recombinant bacterium present
Kpc fimbriae, and further confers on it higher
biofilm-forming activity, indicating that the
Kpc fimbriae may contribute to K. pneumoniae
biofilm formation [5 8] .
●●KPF-28 adhesin
As a polymer of a 28-kDa major fimbrin subunit,
KPF-28 is a long, thin and flexible fimbria that
is 4–5 nm in diameter and 0.5–2 mm in length,
and the structural gene of the KPF-28 major
subunit is located on a transferable R plasmid
encoding the CAZ-5/SHV-4 β-lactamase [59 ] .
The multidrug-resistant K. pneumoniae strains
producing plasmid-encoded CAZ-5/SHV-4 and
KPF-28 represent a epidemic clone of K. pneu-
moniae in the hospitals in Clermont-Ferrand,
France [69]. No KPF-28 expression can be
observed in E. coli transconjugants harboring
the CAZ-5/SHV-4-encoding plasmid alone,
and thus, besides the R plasmid, additional
factor(s) most likely encoded by the K. pneu-
moniae chromosome are required in order to
promote KPF-28 expression [59] . The KPF-28
fimbriae contribute to the adhesion of K. pneu-
moniae to human Caco-2 cell lines, indicating
that the fimbriae may be a colonization factor
within the mammalian intestine [59] .
Outer membrane proteins
●●OmpA
It is evidenced that CPS is necessary but not
sufficient to attenuate airway epithelial cell-
mediated inflammatory responses [35] . OmpA
is one of the major outer membrane proteins of
Gram-negative bacteria, and it is highly con-
served among Enterobacteriaceae. K. pneumo-
niae OmpA, independent of CPS, is important
for preventing the activation of airway epi-
thelial cells via acting on NF-κB-, p38- and
p44/42-dependent pathways and thus contrib-
utes to the attenuation of the airway epithelial
cell-mediated inflammatory response (Figure 1)
[70] . Loss of OmpA makes K. pneumoniae more
susceptible to antimicrobial peptides, but has
no effect on CPS production, which is known
to be responsible for resistance to antimicro-
bial peptides [ 71] . It is thought that OmpA is
involved in the activation of as-yet unknown
systems dedicated to ameliorating the cytotox-
icity of antimicrobial peptides. OmpA also con-
tributes to resistance to phagoyctosis by alveolar
macrophages (Figure 1) [45].
●●Outer membrane porins
K. pneumoniae produces two major outer mem-
brane porins – OmpK35 and OmpK36 – through
which hydrophilic molecules (e.g., nutrients
and cephalosporins/carbapenems) diffuse into
the bacteria [7 2]. In addition, K. pneumoniae
expresses alternative porins, such as KpnO [73]
and OmpK26 [74 ], in order to compensate for
the absence of OmpK35/36. Loss of any of
OmpK36, KpnO or OmpK26 leads to increased
resistance to cephalosporins/carbapenems and
reduced virulence in mouse models of acute
systemic infections, while loss of OmpK35 has
no effect on antibiotic resistance and virulence
[45,7 2–74 ]. Loss of OmpK36 remodels the surface
structure of K. pneumoniae and thereby alters
the binding of phagocytes, leading to increased
susceptibility to phagocytosis and thus an
attenuation in virulence (Figure 1) [45,72] .
●●Eux pumps
K. pneumoniae expresses the efflux pump AcrAB,
which contributes to the export of not only
antibiotics (e.g., quinolones and β-lactams),
but also host-derived antimicrobial agents (e.g.,
the antimicrobial agents present in human
bronchoalveolar lavage fluid and human anti-
microbial peptides; Figure 1), and AcrAB acts as a
determinant of K. pneumoniae resistance to host

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innate immune defenses [75] . The inactivation
of AcrAB not only leads to a multidrug resist-
ance phenotype, but also to a reduced capac-
ity to cause pneumonia in a murine model [75] .
The expression of another K. pneumoniae efflux
pump, namely EefABC, is not related to any
antibiotic resistance phenotype, but it confers
acid tolerance in vitro and high competition
potential in the host GI tract [76].
Iron acquisition
Iron is essential for bacterial growth both in
vitro and in vivo. At least 12 distinct iron uptake
systems can be identified in K. pneumoniae
NTUH-K2044, and they can be assigned into
four major classes: Fe2+ transporter Feo, ABC
transporter, hemophore-based uptake systems
and siderophore-based uptake systems ( Tabl e 1) .
Of these 12 iron uptake systems, two ABC trans-
porters (Kfu [77] and Sit [78]) and three sidero-
phore-based systems (Yersinia high-pathogenicity
island [79, 80] , Iuc [80, 81] and IroA [80] ) have been
shown to be required for the full virulence of
K. pneumoniae. The kfu, high-pathogenicity
island, iuc and iroA regions are highly associated
with hypervirulent K. pneumoniae, thereby repre-
senting horizontally acquired virulence loci dur-
ing the evolution of hypervirulent variants from
opportunistically pathogenic K. pneumoniae [82] .
Bacterial pathogens cope with the scarcity
of iron in their mammalian hosts (in which
iron is bound by various iron-binding proteins)
through synthesizing small iron-scavenging
molecules called hemophores or siderophores.
Enterobacteriaceae pathogens, including K. pneu-
moniae, produce a prototypical siderophore called
enterobactin, which has the highest iron affinity
compared with any other known iron chelators.
Mammals have evolved to secrete lipocalin 2 in
order to sequester enterobactin, leading to the
blocking of siderophore-based iron acquisition
as a part of their innate immune response [83] .
As a countermeasure, a subset of K. pneumoniae
isolates with a tendency to cause clinical respira-
tory or pulmonar y infections produces additional
siderophores, such as yersiniabactin (a phenolate-
type siderophore) and salmochelin (a glycosylated
form of enterobactin), in order to rejuvenate the
siderophore-based iron acquisition pathways,
allowing this pathogen to evade the lipocalin
2-based mechanism of the host innate immune
defense [84,85]. Notably, hypervirulent K. pneumo-
niae secrete quantitatively more and biologically
more active siderophore molecules than classical
K. pneumoniae, delineating an additional mech-
anism by which hypervirulent K. pneumoniae
increases its pathogenic potential [86] .
Nitrogen source utilization
●●Urease
Many gut pathogens, including K. pneumoniae,
can produce cytoplasmic urease in order to hydro-
lyze urea to ammonia and carbon dioxide as a
source of nitrogen for growth. The ureDABCEFG
operon encodes structural subunits (UreA,
UreB and UreC) of the metalloenzyme urease
Table 1. Iron uptake systems in Klebsiella pneumoniae NTUH-K2044.
Category System Substrate Gene name Gene ID
Feo Feo Fe2+ feoABC KP1_5110–KP1_5112
ABC transporter Sit Fe2+ sitABCD KP1_4347–KP1_4350
Kfu Fe3+ kfuABC K P1_19 80– KP1_19 82
Fec Ferric citrate fecBDEA KP1_3247–KP1_3250
Yiu Unknown yiuABC K P1_14 41– KP1_1439
Hemophore based Hmu Hemin/hemoprotein hmuRSTUV KP1_ 4356 –KP1_4362
Siderophore based Fep-Ent Enterobactin fepA-e ntD, fes-entF, fepDGC,
ybdA, fe pB, entCEBA
KP1_1547– KP1_15 46,
KP1_1548–KP1_1549,
KP1_1553–KP1_1551,
KP1_1555, KP1_1556,
KP1_1557–KP1_1560
Fhu Ferrichrome fhuABCD K P1_10 02–KP1_1005
IroA Salmochelin iroN, iroBCD KP1_3609, KP1_3610–K P1_3613
   iroBCDN KP1_p028–KP1_p025
Iuc Aerobactin iucABCD-iutA KP1_p319– KP1_p314
 High-pathogenicity island Yersiniabactin ybtPQXS, ybtA-irp2-irp1-
ybtUTE-fyuA
KP1_3586 –KP1_3583,
KP1_3587–K P1_3593

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future science group
and accessory nickel-binding proteins (UreD,
UreE, UreF and UreG) that are responsible for
the incorporation of nickel ions into the active
site of the urease enzyme [87] . The inactivation
of the K. pneumoniae urease-based metabolism
of urea will impair the growth of this pathogen
in the host GI tract, where urea is abundant [88] .
●●Allantoin metabolism
A 22-kb chromosomal all gene locus responsi-
ble for allantoin metabolism is highly associated
with hypervirulent K. pneumoniae [82] and plays
an important role in K. pneumoniae-induced liver
infection [89] . Primary liver abscesses caused by
hypervirulent K. pneumoniae frequently occur in
diabetes mellitus patients with an increased allan-
toin concentration. The allantoin utilization phe-
notype described in hypervirulent K. pneumoniae
elevates its capability to compete for allantoin as a
nitrogen source in mammalian hosts [8 9].
Conclusion
Ty pic a l K. pneumoniae is an opportunistic
pathogen commonly causing nosocomial infec-
tions, but a subset of hypervirulent serotypes
(including predominantly K1 and K2) due to
increased production of CPS affect previously
healthy persons to cause life-threatening invasive
infections. CPS is the most important virulence
factor of K. pneumoniae, which plays important
roles in resistance to phagocytosis, suppression of
early inflammatory response, resistance to anti-
microbial peptides, and inhibition of DC cell
maturation. Additional K. pneumoniae virulence
factors include LPS, fimbriae, outer membrane
proteins, and determinants for iron acquisition
and nitrogen source utilization.
Future perspective
The current understanding of K. pneumoniae
pathogenesis is mainly derived from studying
individual genes, although global screening
methods, such as signature-tagged mutagenesis
assays [90], have been used for identifying candi-
dates of virulence determination. Huge genetic
variability can be found among different types
of K. pneumoniae, which probably cause dra-
matic differences in pathogenicity [82] . The
regulatory networks governing complex cel-
lular pathways to operate in a concerted man-
ner, which enable the K. pneumoniae lifestyle
in mammalian hosts, are still poorly under-
stood. Genomics and transcriptomics studies
are promising to provide us with deeper under-
standing of the K. pneumoniae–host interac-
tions from a genome-scale view and to iden-
tify numerous candidates of virulence-related
genes for further hypothesis-based functional
characterization.
Financial & competing interests disclosure
Financial support was provided by the Foundation for
Innovative Research Team in Hubei University of Medicine
(2011 CXX01), the Fund for Fostering the National
Natural Science Foundation of Hubei University of
Medicine (2012JPY09), the National Key Program for
Infectious Disease of China (2013ZX10004216) and the
National Basic Research Program of China
(2014CB744400). The authors have no other relevant
affiliations or financial involvement with any organization
or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the
manuscript apart from those disclosed.
No writing assistance was utilized in the production of
this manuscript.
EXECUTivE SUMMARY
Klebsiella pneumoniae-induced infection
● Typ ica l Klebsiella pneumoniae is an opportunistic pathogen that commonly causes nosocomial infections.
● Due to their increased production of capsule polysaccharide, a subset of hypervirulent K.pneumoniae serotypes
(including predominantly K1 and K2) aect previously healthy persons and cause life-threatening invasive infections.
Virulence determinants of K.pneumoniae
● Capsule polysaccharide is the most important virulence factor of K.pneumoniae that plays important roles in resistance
to phagocytosis, suppression of early inammatory responses, resistance to antimicrobial peptides and inhibition of
dendritic cell maturation.
● Additional K.pneumoniae virulence factors include lipopolysaccharide, mbriae, outer membrane proteins and
determinants of iron acquisition and nitrogen source utilization.
● The roles of these virulence factors in survival and immune evasion during infection are discussed in this article.

1079
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Molecular pathogenesis of Klebsiella pneumoniae REviEW
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