Neutralizing human monoclonal antibodies that target the PcrV component of the Type III Secretion System of Pseudomonas aeruginosa act through distinct mechanisms
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Curated by eLife
eLife Assessment
This useful study identifies new monoclonal antibodies produced by cystic fibrosis patients against the Pseudomonas aeruginosa type three secretion system. The evidence supporting the authors' claim is solid. However, in the current version of the manuscript, it is unclear what the benefits of the newly isolated antibodies are with respect to antibodies previously identified using a similar approach. The study will be of interest to those working on developing mAbs against Pseudomonas aeruginosa and also against other pathogens that harbor the T3SS.
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Abstract
Pseudomonas aeruginosa is a major human opportunistic pathogen associated with a high incidence of multi-drug resistance. The antibody-based blockade of P. aeruginosa virulence factors represents a promising alternative strategy to mitigate its infectivity. In this study, we employed single B cell sorting to isolate, from cystic fibrosis patients, human monoclonal antibodies (mAbs) targeting proteins from the P. aeruginosa Type 3 Secretion System (T3SS) and characterized a panel of mAbs directed at PscF and PcrV. Among those, two mAbs, P5B3 and P3D6, that bind to the injectisome tip protein PcrV, exhibited T3SS blocking activity. We solved the crystal structure of the P3D6 Fab-PcrV complex, which revealed that the Ab binds to the C-terminal region of PcrV. Further, we compared the T3SS-blocking activity of three PcrV-targeting mAbs, including two from previous independent studies, using two distinct assays to evaluate pore formation and toxin injection. We conducted a mechanistic and structural analysis of their modes of action through modeling based on the known structure of a functional homolog, SipD from Salmonella typhimurium . The analysis suggests that anti-PcrV mAbs may act through different mechanisms, ranging from preventing PcrV oligomerization to disrupting PcrV’s scaffolding function, thereby inhibiting the assembly and function of the translocon pore. Our findings provide additional evidence that T3SS-targeting Abs, some capable of inhibiting virulence, are elicited in P. aeruginosa -infected patients. The results offer deeper insights into PcrV recognition by mAbs and their associated mechanisms of action, helping to identify which Abs are more likely to be therapeutically useful based on their mode of action and potency. This paves the way for developing effective alternatives to traditional antibiotics in the fight against this resilient pathogen.
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eLife Assessment
This useful study identifies new monoclonal antibodies produced by cystic fibrosis patients against the Pseudomonas aeruginosa type three secretion system. The evidence supporting the authors' claim is solid. However, in the current version of the manuscript, it is unclear what the benefits of the newly isolated antibodies are with respect to antibodies previously identified using a similar approach. The study will be of interest to those working on developing mAbs against Pseudomonas aeruginosa and also against other pathogens that harbor the T3SS.
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Reviewer #1 (Public review):
Summary:
Desveaux et al. describe human mAbs targeting protein from the Pseudomonas aeruginosa T3SS, discovered by employing single cell B cell sorting from cystic fibrosis patients. The mAbs were directed at the proteins PscF and PcrV. They particularly focused on two mAbs binding the T3SS with the potential of blocking activity. The supplemented biochemical analysis was crystal structures of P3D6 Fab complex. They also compared the blocking activity with mAbs that were described in previous studies, using an assay that evaluated the toxin injection. They conducted mechanistic structure analysis and found that these mAbs might act through different mechanisms by preventing PcrV oligomerization and disrupting PcrVs scaffolding function.
Strengths:
The antibiotic resistance crisis requires the development of …
Reviewer #1 (Public review):
Summary:
Desveaux et al. describe human mAbs targeting protein from the Pseudomonas aeruginosa T3SS, discovered by employing single cell B cell sorting from cystic fibrosis patients. The mAbs were directed at the proteins PscF and PcrV. They particularly focused on two mAbs binding the T3SS with the potential of blocking activity. The supplemented biochemical analysis was crystal structures of P3D6 Fab complex. They also compared the blocking activity with mAbs that were described in previous studies, using an assay that evaluated the toxin injection. They conducted mechanistic structure analysis and found that these mAbs might act through different mechanisms by preventing PcrV oligomerization and disrupting PcrVs scaffolding function.
Strengths:
The antibiotic resistance crisis requires the development of new solutions to treat infections caused by MDR bacteria. The development of antibacterial mAbs holds great potential. In that context, this report is important as it paves the way for the development of additional mAbs targeting various pathogens that harbor the T3SS. In this report, the authors present a comparative study of their discovered mAbs vs. a commercial mAb currently in clinical testing resulting in valuable data with applicative implications. The authors investigated the mechanism of action of the mAbs using advanced methods and assays for the characterization of antibody and antigen interaction, underlining the effort to determine the discovered mAbs suitability for downstream application.
Weaknesses:
Although the information presented in this manuscript is important, previous reports regarding other T3SS structures complexed with antibodies, reduce the novelty of this report. Nevertheless, we provide several comments that may help to improve the report. The structural analysis of the presented mAbs is incomplete and unfortunately, the authors did not address any developability assessment. With such vital information missing, it is unclear if the proposed antibodies are suited for diagnostic or therapeutic usage. This vastly reduces the importance of the possibly great potential of the authors' findings. Moreover, the structural information does not include the interacting regions on the mAb which may impede the optimization of the mAb if it is required to improve its affinity.
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Reviewer #2 (Public review):
Summary:
Desveaux et al. performed Elisa and translocation assays to identify among 34 cystic fibrosis patients which ones produced antibodies against P. aeruginosa type three secretion system (T3SS). The authors were especially interested in antibodies against PcrV and PcsF, two key components of the T3SS. The authors leveraged their binding assays and flow cytometry to isolate individual B cells from the two most promising sera, and then obtained monoclonal antibodies for the proteins of interest. Among the tested monoclonal antibodies, P3D6 and P5B3 emerged as the best candidates due to their inhibitory effect on the ExoS-Bla translocation marker (with 24% and 94% inhibition, respectively). The authors then showed that P5B3 binds to the five most common variants of PcrV, while P3D6 seems to recognize only …
Reviewer #2 (Public review):
Summary:
Desveaux et al. performed Elisa and translocation assays to identify among 34 cystic fibrosis patients which ones produced antibodies against P. aeruginosa type three secretion system (T3SS). The authors were especially interested in antibodies against PcrV and PcsF, two key components of the T3SS. The authors leveraged their binding assays and flow cytometry to isolate individual B cells from the two most promising sera, and then obtained monoclonal antibodies for the proteins of interest. Among the tested monoclonal antibodies, P3D6 and P5B3 emerged as the best candidates due to their inhibitory effect on the ExoS-Bla translocation marker (with 24% and 94% inhibition, respectively). The authors then showed that P5B3 binds to the five most common variants of PcrV, while P3D6 seems to recognize only one variant. Furthermore, the authors showed that P3D6 inhibits translocon formation, measured as cell death of J774 macrophages. To get insights into the P3D6-PcrV interaction, the authors defined the crystal structure of the P3D6-PcrV complex. Finally, the authors compared their new antibodies with two previous ones (i.e., MEDI3902 and 30-B8).
Strengths:
(1) The article is well written.
(2) The authors used complementary assays to evaluate the protective effect of candidate monoclonal antibodies.
(3) The authors offered crystal structure with insights into the P3D6 antibody-T3SS interaction (e.g., interactions with monomer vs pentamers).
(4) The authors put their results in context by comparing their antibodies with respect to previous ones.
Weaknesses:
(1) The authors used a similar workflow to the one previously reported in Simonis et al. 2023 (antibodies from cystic fibrosis patients that included B cell isolation, antibody-PcrV interaction modeling, etc.) but the authors do not clearly explain how their work and findings differentiate from previous work.
(2) Although new antibodies against P. aerugisona T3SS expand the potential space of antibody-based therapies, it is unclear if P3D6 or P5B3 are better than previous antibodies. In fact, in the discussion section authors suggested that the 30-B8 antibody seems to be the most effective of the tested antibodies.
(3) The authors should explain better which of the two antibodies they have discovered would be better suited for follow-up studies. It is confusing that the authors focused the last sections of the manuscript on P3D6 despite P3D6 having a much lower ExoS-Bla inhibition effect than P5B3 and the limitation in the PcrV variant that P3D6 seems to recognize. A better description of this comparison and the criteria to select among candidate antibodies would help readers identify the main messages of the paper.
(4) This work could strongly benefit from two additional experiments:
a) In vivo experiments: experiments in animal models could offer a more comprehensive picture of the potential of the identified monoclonal antibodies. Additionally, this could help to answer a naïve question: why do the patients that have the antibodies still have chronic P. aeruginosa infections?
b) Multi-antibody T3SS assays (i.e., a combination of two or more monoclonal antibodies evaluated with the same assays used for characterization of single ones). This could explore the synergistic effects of combinatorial therapies that could address some of the limitations of individual antibodies. -
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