Pathogenesis and Drug Development Caitlin

pathogensReview
Adhesive Pili in UTI Pathogenesis and
Drug Development
Caitlin N. Spaulding 1 and Scott J. Hultgren 1,2,*
1 Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110,
USA; [e-mail]
2 Center for Women’s Infectious Disease Research, Washington University School of Medicine, St. Louis,
MO 63110, USA
* Correspondence: [e-mail]; Tel.: [phone]
Academic Editor: Lawrence S. Young
Received: 16 July 2015; Accepted: 7 March 2016; Published: 15 March 2016
Abstract: Urinary tract infections (UTIs) are one of the most common bacterial infections, affecting
150 million people each year worldwide. High recurrence rates and increasing antimicrobial resistance
among uropathogens are making it imperative to develop alternative strategies for the treatment and
prevention of this common infection. In this Review, we discuss how understanding the: (i) molecular
and biophysical basis of host-pathogen interactions; (ii) consequences of the molecular cross-talk at the
host pathogen interface in terms of disease progression; and (iii) pathophysiology of UTIs is leading
to efforts to translate this knowledge into novel therapeutics to treat and prevent these infections.
Keywords: UTI; rUTI; CAUTI; pili; UPEC; chaperone-usher pathway (CUP) pili; Enterococcus; vaccine;
antibiotic-resistance
1. Introduction
Urinary tract infections (UTIs) can be acquired in the community or hospital setting and are one
of the most common bacterial infections that occur, affecting more than 150 million people worldwide
each year [1–3 ]. UTI is clinically divided into two major infections, characterized by the localization
of the bacteria in the urinary tract, cystitis and pyelonephritis. Cystitis, or lower UTI, is infection of
the bladder. Once in the bladder, bacteria can ascend the ureters and colonize the kidneys, causing
pyelonephritis or upper UTI. While the incidence of pyelonephritis is fairly low (~0.3%–0.6%) it is
particularly dangerous as uncontrolled bacterial infection can spread to the bloodstream, causing
sepsis (which occurs in ~2% of pyelonephritis cases) [4 ,5 ]. UTIs are also categorized as uncomplicated
or complicated infections. Complicated UTI occurs in patients with: (i) functional or structural urinary
tract abnormalities; (ii) renal failure; (iii) immunosuppression; (iv) pregnancy; and/or (v) foreign
bodies, such as indwelling catheters, placed within their urinary tract [ 6– 8]. Catheter-associated UTIs
(CAUTI), make up 70%–80% of all complicated UTI and are the most common type of nosocomial
infection [4 ,9]. CAUTI are of particular concern as they result in high morbidity, increased mortality
and are the most common cause of secondary sepsis in hospital patients. While complicated UTI
affects individuals of both genders, uncomplicated UTI primarily affects otherwise healthy women [10 ].
Pyelonephritis often occurs in healthy, non-pregnant women but can be categorized as a complicated
UTI because of the potential of developing a blood stream infection. For women, the lifetime risk of
developing an uncomplicated UTI approaches 60% [5]. Of these women who experience an initial
UTI, 20%–30% will go on to experience a recurrent infection (rUTI) within 4–6 months, despite
receiving appropriate antibiotic therapy [ 5, 11]. To combat rUTI, these women are treated with frequent
antibiotic therapy often to be taken at the time that symptoms arise or immediately following sexual
intercourse [6 ]. However, a subset of these women will continue to experience rUTI as frequently as
Pathogens 2016, 5, 30; doi:10.3390/pathogens5010030 www.mdpi.com/journal/pathogens

Pathogens 2016, 5, 30 2 of 18
six or more times a year [12 ]. Due to its prevalence and high rates of recurrence, UTI is associated
with significant economic costs. The financial burden of UTI in the United States, which reflects
both direct medical costs and indirect costs such as lost work output and wages, is an estimated
$5 billion annually [5]. These infections also result in significant patient morbidity resulting in serious
deterioration in quality of life including: pain, discomfort, disruption of daily activities, and few
treatment options other than long-term antibiotic prophylaxis [5,11,13].
While many bacterial organisms cause UTI, the most common causative agent of both
uncomplicated and complicated UTI is the gram-negative pathogen uropathogenic Escherichia coli
(E. coli) (UPEC). UPEC are responsible for 80%–90% of all uncomplicated UTI and approximately
65% of complicated UTIs [ 5 ,14 ,15 ]. Gram-positive Enterococcus species are the second leading cause
of complicated UTI (11%) and the third leading cause of uncomplicated UTI (5%) [ 15 ]. The source
population of UPEC and Enterococcus that lead to UTI is thought to be the gastrointestinal tract, where
they can reside as either commensal or transient members of the gut microbiota [ 11 ,16 ,17 ]. When
present in the gut, UPEC or Enterococcus spp. can be shed in the feces, inoculating peri-urethral
or vaginal areas, and are subsequently introduced into the urinary tract during periods of physical
manipulation such as during sexual activity or catheterization (Figure 1A) [18 ]. Upon entering the
bladder, uropathogens must bind to an available epithelial receptor and/or, if present, abiotic-surface
to establish and maintain colonization. UPEC and enterococcal species both accomplish this through
the expression of distinctive adhesive pili on their surface. After creating a foothold in the bladder,
uropathogens employ a myriad of additional virulence factors to establish bladder colonization in
the face of an active immune response, micturition, and rapid epithelial cell exfoliation. Historically,
antibiotics have been used, very successfully, to treat patients with UTI. However, the rise of single
and multi-drug resistant uropathogens as well as high rates of recurrence in women infected with
both antibiotic sensitive and drug-resistant uropathogens has become a major concern, highlighting
the need to develop alternative strategies to treat patients with UTI and CAUTI. In this review, we
will discuss the role of adhesive pili during UTI or CAUTI. Here we will focus mainly on UTI and
CAUTI caused by UPEC and Enterococcus spp. due to the high prevalence of these pathogens in
community-acquired and nosocomial infections. We will also explore the development of alternative,
non-antibiotic treatment strategies that target adhesive pili in order to prevent UPEC and Enterococcus
spp. from initiating infection and thus causing disease.

Pathogens 2016, 5, 30 3 of 18Pathogens 2016, 5, 30
Figure 1. Uropathogenic E. coli (UPEC ) pathogenic cascade during cystitis. (A) UPEC residing in the
gut are shed in the feces and colonize the peri-urethral and vaginal areas before ascending into the
bladder. Upon accessing the bladder, UPEC adhere to the surface of superficial facet cells that line the
bladder lumen in a type 1 pili dependent manner (B). Adherent bacteria invade into the facet cells
and are either expelled back into the lumen by the cell in a TLR-4 dependent manner [19] (C) or escape
from the endocytic vesicle into the cytoplasm (D). Upon invasion, bacteria replicate in the cytoplasm
forming intracellular bacterial communities (IBCs) (E). One host mechanism of defense against
intracellular UPEC is the shedding of urothelial cells into the urine (F), which reduces the overall
number of UPEC in the bladder. During the late stages of IBC formation, filamentous bacteria
dissociate from the IBC, burst out of the cell and back into the bladder lumen where they remain or
can invade an adjacent facet cell (G). There are two potential outcomes of infection: chronic cystitis or
resolution of infection. Uncontrolled bacterial replication in the urine occurs in mice that develop
chronic cystitis (H). In mice that resolve infection, small pockets of bacteria, termed quiescent
intracellular reservoirs (QIRs), form and reside in the underlying urothelium and may seed future rUTI (I).
2. The Role of Chaperone-Usher Pathway (CUP) Pili in UPEC Mediated UTI
2.1. CUP Pilus Assembly Mechanisms
Upon entering the bladder, UPEC must first adhere to the bladder epithelium, also referred to
as the urothelium, or risk clearance during urine voiding. Recognition and attachment to host and
environmental surfaces is mediated through the expression of non-flagellar, adhesive, extracellular
fibers, called pili that bind to receptors present on the host cell surface. In UPEC, many of these
adhesive pili belong to a large, conserved family of pili called the chaperone-usher pathway (CUP)
pili [20]. CUP pili are assembled by the corresponding chaperone-usher machinery, which are
encoded by operons that contain all the dedicated genetic information necessary to assemble a mature
pilus: an outer-membrane pore-forming usher protein, a periplasmic chaperone protein, pilus
subunits, and in most cases, a tip adhesin protein. The first crystal structure of a CUP chaperone, PapD,
which is involved in the assembly of P pili, revealed that it consists of two-immunoglobulin (Ig)
domains [21]. Two key amino acid residues, R8 and K112, present in the cleft of the chaperone were
subsequently identified as the active site of the protein [22]. Unlike the chaperone, pilus subunits are
composed of an incomplete Ig fold, which lacks the C-terminal beta strand and requires the assistance
of the dedicated chaperone for folding and stability (Figure 2B,D). Chaperone-assisted folding occurs
by a reaction termed donor strand complementation (DSC) in which conserved alternating exposed
Figure 1. Uropathogenic E. coli (UPEC ) pathogenic cascade during cystitis. (A) UPEC residing in the
gut are shed in the feces and colonize the peri-urethral and vaginal areas before ascending into the
bladder. Upon accessing the bladder, UPEC adhere to the surface of superficial facet cells that line the
bladder lumen in a type 1 pili dependent manner (B). Adherent bacteria invade into the facet cells and
are either expelled back into the lumen by the cell in a TLR-4 dependent manner [19 ] (C) or escape from
the endocytic vesicle into the cytoplasm (D). Upon invasion, bacteria replicate in the cytoplasm forming
intracellular bacterial communities (IBCs) (E). One host mechanism of defense against intracellular
UPEC is the shedding of urothelial cells into the urine (F), which reduces the overall number of UPEC
in the bladder. During the late stages of IBC formation, filamentous bacteria dissociate from the IBC,
burst out of the cell and back into the bladder lumen where they remain or can invade an adjacent
facet cell (G). There are two potential outcomes of infection: chronic cystitis or resolution of infection.
Uncontrolled bacterial replication in the urine occurs in mice that develop chronic cystitis (H). In mice
that resolve infection, small pockets of bacteria, termed quiescent intracellular reservoirs (QIRs), form
and reside in the underlying urothelium and may seed future rUTI (I).
2. The Role of Chaperone-Usher Pathway (CUP) Pili in UPEC Mediated UTI
2.1. CUP Pilus Assembly Mechanisms
Upon entering the bladder, UPEC must first adhere to the bladder epithelium, also referred to
as the urothelium, or risk clearance during urine voiding. Recognition and attachment to host and
environmental surfaces is mediated through the expression of non-flagellar, adhesive, extracellular
fibers, called pili that bind to receptors present on the host cell surface. In UPEC, many of these
adhesive pili belong to a large, conserved family of pili called the chaperone-usher pathway (CUP)
pili [20]. CUP pili are assembled by the corresponding chaperone-usher machinery, which are encoded
by operons that contain all the dedicated genetic information necessary to assemble a mature pilus:
an outer-membrane pore-forming usher protein, a periplasmic chaperone protein, pilus subunits,
and in most cases, a tip adhesin protein. The first crystal structure of a CUP chaperone, PapD,
which is involved in the assembly of P pili, revealed that it consists of two-immunoglobulin (Ig)
domains [21 ]. Two key amino acid residues, R8 and K112, present in the cleft of the chaperone were
subsequently identified as the active site of the protein [22 ]. Unlike the chaperone, pilus subunits
are composed of an incomplete Ig fold, which lacks the C-terminal beta strand and requires the

Pathogens 2016, 5, 30 4 of 18
assistance of the dedicated chaperone for folding and stability (Figure 2B,D). Chaperone-assisted
folding occurs by a reaction termed donor strand complementation (DSC) in which conserved
alternating exposed hydrophobic residues on the chaperone’s G1 strand are buried in complementary
pockets in the pilus subunit, allowing for the completion of the subunits Ig fold (Figure 2C) [ 23, 24].
This interaction allows pilus subunits to fold into a primed, high-energy state in complex with the
chaperone, ultimately allowing the subunits to be targeted to the outer membrane usher (Figure 2E).
The usher, a gated channel, is made up of five functional domains: a 24 stranded transmembrane
β-barrel translocation domain (TD), a β-sandwich plug (PLUG) that resides in the pore of the TD in the
apo-usher, an N-terminal periplasmic domain (NTD) and two C-terminal periplasmic domains (CTD1
and 2) [25–29]. The usher catalyzes pilus assembly by driving subunit polymerization in a concerted
reaction termed donor strand exchange (DSE) (Figure 2E,F) [30 – 33]. Pilus subunits, excluding the
adhesin, encode an N-terminal extension (Nte) comprised of conserved alternating hydrophobic
residues. DSE occurs when the chaperone is displaced and an incoming subunit’s Nte zips into the
previously chaperone-bound groove of a nascently incorporated subunit at the growing terminus of
the pilus. This “zip-in-zip-out mechanism” allows for the final folding of the pilus subunit, such that
every subunit in the pilus completes the Ig fold of its neighbor (Figure 2F).Pathogens 2016, 5, 30
hydrophobic residues on the chaperone’s G1 strand are buried in complementary pockets in the pilus
subunit, allowing for the completion of the subunits Ig fold (Figure 2C) [23,24]. This interaction allows
pilus subunits to fold into a primed, high-energy state in complex with the chaperone, ultimately
allowing the subunits to be targeted to the outer membrane usher (Figure 2E). The usher, a gated
channel, is made up of five functional domains: a 24 stranded transmembrane β-barrel translocation
domain (TD), a β-sandwich plug (PLUG) that resides in the pore of the TD in the apo-usher, an N-
terminal periplasmic domain (NTD) and two C-terminal periplasmic domains (CTD1 and 2) [25–29].
The usher catalyzes pilus assembly by driving subunit polymerization in a concerted reaction termed
donor strand exchange (DSE) (Figure 2E,F) [30–33]. Pilus subunits, excluding the adhesin, encode an
N-terminal extension (Nte) comprised of conserved alternating hydrophobic residues. DSE occurs
when the chaperone is displaced and an incoming subunit’s Nte zips into the previously chaperone-
bound groove of a nascently incorporated subunit at the growing terminus of the pilus. This “zip-in-
zip-out mechanism” allows for the final folding of the pilus subunit, such that every subunit in the
pilus completes the Ig fold of its neighbor (Figure 2F).
Figure 2. Mannosides and Pilicides prevent uropathogenic E. coli (UPEC) UTI by targeting the
function or formation of type 1 pili. (A,G) Unfolded pilus subunits are secreted to the periplasm by
the Sec apparatus. (B,H) Upon entering the periplasm, unfolded subunits immediately interact with
the cognate chaperone (FimC). Subunits have an incomplete Ig-like fold which must be completed in
order to properly fold. In a process called donor strand complementation (DSC) FimC donates its G1
β-stand to the subunit, stabilizing it (C,I). Subunits that do not interact with FimC are unable to fold
correctly and are degraded (D). The chaperone then delivers the subunit to the outer member usher,
FimD (E). Upon reaching FimD the subunit is assembled into the maturing pilus via donor stand
exchange (DSE) with the adjacent pilus subunit (F). Mannosides prevent type 1 pilus function by
binding, in an irreversible manner, to FimH and therefore prevent the interaction of FimH and
mannose on the bladder surface (G). Pilicide works by halting pilus assembly. These molecules enter
the periplasm (J) and bind to the pilus chaperone, halting assembly (K).
2.2. The Role of Type 1 Pili during Uncomplicated UTI
Figure 2. Mannosides and Pilicides prevent uropathogenic E. coli (UPEC) UTI by targeting the function
or formation of type 1 pili. (A,G) Unfolded pilus subunits are secreted to the periplasm by the Sec
apparatus. (B,H) Upon entering the periplasm, unfolded subunits immediately interact with the
cognate chaperone (FimC). Subunits have an incomplete Ig-like fold which must be completed in
order to properly fold. In a process called donor strand complementation (DSC) FimC donates its
G1 β-stand to the subunit, stabilizing it (C,I). Subunits that do not interact with FimC are unable to
fold correctly and are degraded (D). The chaperone then delivers the subunit to the outer member
usher, FimD (E). Upon reaching FimD the subunit is assembled into the maturing pilus via donor
stand exchange (DSE) with the adjacent pilus subunit (F). Mannosides prevent type 1 pilus function
by binding, in an irreversible manner, to FimH and therefore prevent the interaction of FimH and
mannose on the bladder surface (G). Pilicide works by halting pilus assembly. These molecules enter
the periplasm (J) and bind to the pilus chaperone, halting assembly (K).