1 | Introduction
The emergence of multiple-drug resistant (MDR) bacterial strains stems
largely from the extensive, and sometimes inappropriate, usage of
antibiotics in the community and in agriculture, as this misuse has
exerted a strong selective pressure on bacteria to develop resistance
mechanisms against various antibiotics (Laxminarayan et al., 2013;
Laxminarayan & Heymann, 2012). In turn, the implications of the
increasing numbers of MDR bacterial infections in the clinic, in the
community, and in agriculture are constituting a growing global public
health concern (Ventola, 2015): MDR bacterial infections are harder to
treat and are associated with higher medical costs than
antibiotic-sensitive infections, and, perhaps more importantly, there is
a significant risk that MDR mechanisms will be spread to other bacterial
strains (Jiang et al., 2017). A parallel public health concern is that
the development and approval of new antibiotics has not kept pace with
the rising rates of morbidity and mortality due to bacterial infections,
giving rise to a predicted annual death rate of 10 million people by
2050 due to resistance to antimicrobials (O’Neill, 2016). The lack of
progress in the development of antibiotics may be attributed not only to
the limited discovery of suitable molecular targets, but also to the
absence of significant investment on the part of large pharmaceutical
companies (Munguia & Nizet, 2017). Yet another health concern lies in
the accumulating evidence that broad-spectrum antibiotics have a
detrimental effect on the native microbiome (Nizet, 2015), which is
regarded to play a beneficial role in human and animal health: It is
currently held that impairment of the microbiome can lead to long-term
diarrhea, diabetes, obesity and immune defects (Cox & Blaser, 2015;
Leslie & Young, 2015; Modi, Collins, & Relman, 2014; Theriot et al.,
2014). Taken together, the above factors call for the development and
implementation of new therapeutic strategies that specifically target
bacterial virulence mechanisms. It is likely that such strategies would
apply a milder evolutionary pressure and specifically harm bacterial
pathogens while sparing the beneficial microbiome.
A particularly promising means for providing both therapeutic strategies
targeting bacterial virulence and diagnostic applications lies in
monoclonal antibodies (mAbs) targeted against pathogen-specific
antigens. It has, for example, been demonstrated that a number of mAbs
exhibit high efficacy as protein blockers, especially against bacterial
toxins (Dickey, Cheung, & Otto, 2017), and as diagnostic agents for the
detection of bacteria (Guttikonda, Tang, Yang, Armstrong, & Suresh,
2007). In keeping with this line of thought, recent advances in the
discovery, engineering, production, and clinical development of mAbs
indicate their potential both in the treatment of infectious diseases
and in the design of rapid diagnostics. The use of mAbs as
anti-bacterial agents, either alone or in combination with antibiotics,
can compensate for the inherent limitations of currently available
antibiotics, namely, their inability to clear pro-inflammatory bacterial
components from the circulation or to promote opsonization. Furthermore,
by virtue of their specificity, anti-bacterial mAbs can exclusively
target the pathogen, thereby sparing the microbiome.
Pivotal to the efficiency of controlling antibiotic resistance is the
ability to provide rapid and accurate surveillance and diagnosis (Levin,
Baquero, & Johnsen, 2014), as is embodied in the WHO One Health concept
for addressing the MDR crisis (Hernando-Amado, Coque, Baquero, &
Martinez, 2019). In this regard, the major disadvantages of currently
available laboratory-based diagnostics for the detection of bacterial
infections are long processing times, low sensitivity and specificity,
and/or the need for specialized equipment that is expensive and requires
highly trained personnel (Fournier et al., 2013). Among the
laboratory-based methods currently in use for bacterial diagnosis,
bacterial culturing is probably the most frequently used method, but it
is relatively slow and it is limited to bacteria that can be cultured in
the laboratory. Other methods are based on immunoassays [including
enzyme-linked immunosorbent assays (ELISA) and agglutination assays]
that detect surface bacterial antigens and on genetic analyses that
allow rapid identification of bacterial strains by employing a
polymerase chain reaction (PCR). The latter methods are the most
sensitive, but even they may yield false-positive results and they may
overlook genetically mutated strains. A possible solution was thought to
lie in rapid real-time PCR or mass-spectroscopy techniques, but these,
too, require specialized equipment and reagents and trained personnel
(Burnham & Carroll, 2013; Croxen et al., 2013; Espy et al., 2006). The
above-described obstacles may culminate in misdiagnosed or belatedly
diagnosed bacterial infections and the misuse of antibiotics, and hence,
ultimately, in the exacerbation of the antibiotic resistance crisis.
There is, thus, an imperative need for more rapid, cost-effective, and
sensitive assays that can identify infective agents at the point of
care, without the requirement for multistep processing—a need that
could, for example, be met by antibody-based biosensors.
The above considerations are particularly relevant to the diagnosis and
treatment of Gram-negative bacterial pathogens, such asEscherichia coli , and species of Salmonella, Shigella ,Yersinia, and Pseudomonas , which cause serious diseases,
ranging from lethal diarrhea to sepsis, leading to millions of deaths
annually (Croxen et al., 2013; Dekker & Frank, 2015; Khalil et al.,
2018). An essential component common to these bacterial pathogens is a
syringe-like protein complex, termed the type 3 secretion system (T3SS),
which is responsible for injecting virulence factors from the bacterial
cytoplasm directly into the human host cell (Kaper, Nataro, & Mobley,
2004). This T3SS complex is essential for bacterial virulence, as the
injected proteins (effectors) manipulate key intracellular host pathways
(e.g., cell cycle, immune response, cytoskeletal organization, metabolic
processes and intracellular trafficking) that ultimately promote
bacterial replication and transmission (Bhavsar, Guttman, & Finlay,
2007; Cornelis, 2006). The concept underlying this study – and others
like it – is that the TSS system therefore constitutes a potential
anti-bacterial target, particularly since it is known that many
bacterial strains deleted of a single T3SS gene become non-virulent
(Coburn, Sekirov, & Finlay, 2007; Deng et al., 2017; Deng et al.,
2004).
In the current study, we focused on the T3SS of enteropathogenicE. coli (EPEC), the causative agent of infantile diarrhea (Croxen
et al., 2013). The EPEC T3SS comprises more than 20 proteins, three of
which – EspA, EspB, and EspD – are highly exposed to the extracellular
environment. EspA forms a long filamentous structure that bridges
between the bacterial and host cells, and EspB and EspD together form a
translocator pore complex that facilitates the passage of effectors
across the host plasma membrane. Of these three proteins, we chose to
target EspB by developing a mAb with high EspB affinity and specificity
for therapeutic and/or diagnostic applications. Our rationale for
pinpointing EspB derived from previous findings that a bacterial strain
deleted of the espB gene was unable to infect host cells (Wolff,
Nisan, Hanski, Frankel, & Rosenshine, 1998) and that a similar mutation
in the related murine pathogen, Citrobacter rodentium , was
non-virulent in mice (Deng et al., 2004). We thus report the development
and characterization of mAb-EspB-B7 in a novel application against the
T3SS of EPEC. The high specificity and affinity of mAb-EspB-B7 towards
EspB and its high stability under a variety of conditions make this
antibody an excellent candidate for future development as an
antibacterial drug or as an integral component of a diagnostic
apparatus.