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.