Many bacterial pathogens utilize a specialized secretion system, termed type III secretion system (T3SS), to translocate effector proteins into host cells and establish bacterial infection. The T3SS is anchored within the bacterial membranes and contains a long needle/filament that extends toward the host-cell and forms, at its distal end, a pore complex within the host membrane. The T3SS pore complex consists of two bacterial proteins, termed SctB and SctE, which have conflicting targeting indications; a signal sequence that targets to secretion to the extracellular environment via the T3SS, and transmembrane domains (TMDs) that target to membrane localization. In this study, we investigate whether the TMD sequences of SctB and SctE have special features that differentiate them from classical TMDs and allow them to escape bacterial membrane integration. For this purpose, we exchanged the SctB and SctE native TMDs for alternative hydrophobic sequences and found that the TMD sequences of SctB and SctE dictate membrane destination (bacterial versus host membrane). Moreover, we examined the role of the SctB TMD sequence in the activity of the full-length protein, post secretion, and found that the TMD does not serve only as a hydrophobic segment, but is also involved in the ability of the protein to translocate itself and other proteins into and across the host cell membrane.

It is predicted that the antibiotic resistance crisis will result in an annual death rate of 10 million people by the year 2050. To grapple with the challenges of the impending crisis, there is an urgent need for novel and rapid diagnostic tools. In this study, we developed a novel monoclonal antibodynamed mAb-EspB-B7that targets the EspB protein, a component within the bacterial type 3 secretion system (T3SS), which is mainly expressed in Gram-negative pathogens and is essential for bacterial infectivity. We found that mAb-EspB-B7 has high affinity and specificity toward recombinant and native EspB proteins; is stable over a range of pH levels, temperatures, and salt concentrations; and retains its functionality in human serum. We identified the epitope for mAb-EspB-B7 and validated it by competitive enzyme-linked immunosorbent assay (ELISA). Since this epitope is conserved across several T3SS-harboring pathogens, mAb-EspB-B7 holds great potential for development as an active component in precise and rapid diagnostic tools that can differentiate between commensal and pathogenic bacterial strains. To this end, we integrated the well-characterized monoclonal antibody into an electrochemical biosensor and demonstrated its high specificity and sensitivity capabilities in detecting pathogenic bacterial T3SS-associated antigens as well as intact bacteria. We foresee that in the near future it will be possible to design and develop a point-of-care biosensor with multiplexing capabilities for the detection of a panel of pathogenic bacteria.

It is predicted that failure to address the antibiotic-resistance crisis will result in an annual death rate of 10 million people by the year 2050. To grapple with the challenges of the impending crisis, there is an urgent need for novel anti-bacterial agents and rapid diagnostic tools. Here, we developed a novel monoclonal antibody-known as mAb-EspB-B7-that targets EspB, a component within the bacterial type 3 secretion system (T3SS), which is mainly expressed in Gram-negative pathogens and is essential for bacterial infectivity. We found that mAb-EspB-B7 has high affinity and specificity towards recombinant and native EspB proteins, is stable over a range of pH levels, temperatures and salt concentrations, and retains its functionality in human serum. We identified the epitope for mAb-EspB-B7 and validated it by competitive ELISA. Since this epitope is conserved across several T3SS-harboring pathogens, mAb-EspB-B7 holds great potential for development as an anti-bacterial agent or as the active component in precise and rapid diagnostic tools.

IntroductionEnteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen and one of the major causes of gastrointestinal illness in developing countries. EPEC, similar to many other Gram-negative bacterial pathogens, possesses essential virulence machinery called the type III secretion system (T3SS) that enables the injection of effector proteins from the bacteria into the host cytoplasm. Of these, the translocated intimin receptor (Tir) is the first effector to be injected, and its activity is essential for the formation of attaching and effacing lesions, the hallmark of EPEC colonization. Tir belongs to a unique group of transmembrane domain (TMD)-containing secreted proteins, which have two conflicting destination indications, one for bacterial membrane integration and another for protein secretion. In this study, we examined whether TMDs participate in the secretion, translocation, and function of Tir in host cells.MethodsWe created Tir TMD variants with the original or alternative TMD sequence.ResultsWe found that the C-terminal TMD of Tir (TMD2) is critical for the ability of Tir to escape integration into the bacterial membrane. However, the TMD sequence was not by itself sufficient and its effect was context-dependent. Moreover, the N-terminal TMD of Tir (TMD1) was important for the postsecretion function of Tir at the host cell.DiscussionTaken together, our study further supports the hypothesis that the TMD sequences of translocated proteins encode information crucial for protein secretion and their postsecretion function.