Engineered probiotic Lactobacillus plantarum WCSF I for monitoring and treatment of Staphylococcus aureus infection

ABSTRACT Lactobacillus plantarum is one of the most thoroughly researched species of the genus Lactobacillus, which possesses the characteristics of easy genetic transformation, high-density growth, and high intestinal tract survival. L. plantarum has been proven to play a potential role as a probiotic delivery vector. Staphylococcus aureus is a common Gram-positive pathogenic bacterium. It uses an autoinducer peptide (AIP) produced by its Agr quorum-sensing (AgrQS) system to sense the population density. Using the quorum-sensing mechanism exclusive to S. aureus, we constructed an AgrQS system in L. plantarum WCSF induction and killing modules based on AIP sensing and regulation so that L. plantarum could effectively eliminate S. aureus when detecting exogenous AIP at nanomolar concentrations. By optimizing the expression strength of the two-component system AgrAC using different L. plantarum-derived promoters and replacing the core promoter of the AgrA-activating promoter, the activation strength of AgrQS increased from the initial 1.2-fold to 5.3-fold. By introducing the signal peptide N20-guided lysostaphin aureus protein, engineered L. plantarum was able to effectively control the release of lysostaphin aureus protein and inhibit the growth of S. aureus. For the first time, engineered L. plantarum can detect and treat S. aureus infection, laying the groundwork for the future development of engineered probiotics for the monitoring and therapy of intestinal pathogens. IMPORTANCE Bacterial infection and the emergence of drug-resistant strains are major problems in clinical treatment. Staphylococcus aureus, which typically infects the skin and blood of animals, is also a potential intestinal pathogen that needs to be addressed by the emergence of a new treatment approach. Probiotic therapy is the most likely alternative to antibiotic therapy to solve the problem of bacterial drug resistance in clinical practice. In this study, the engineered Lactobacillus plantarum can not only sense the signal AIP to detect S. aureus but also kill S. aureus by secreting the lysostaphin enzyme. Our strategy employed an Agr quorum-sensing genetic circuit to simultaneously detect and treat pathogenic bacteria, which provided a theoretical possibility for solving practical clinical bacterial infection cases in the future.


Reviewer comments:
Reviewer #1 (Comments for the Author): Qian Wang et al. achieved significant success in inhibiting the growth of S. aureus by introducing a signal peptide N20 to guide the release of lysostaphin aureus protein in engineered L. plantarum.By utilizing various promoters and switching the core promoter for the AgrA-activating promoter, they were able to significantly increase the production of lysostaphin aureus protein.This increase in production levels was a critical factor in the successful inhibition of S. aureus growth.While the manuscript's topic is important and the results are promising, the analysis and writing require significant improvements.To enhance the manuscript's impact and value, the author should consider providing more detailed explanations for their experimental design and findings.Expanding upon their methods and clearly defining the significance of their results would help readers better understand the research and its potential applications.
Major comments: 1.To further support their findings, the authors should consider conducting an HPLC analysis of both synthetic AIP and AIP secreted by S. aureus simultaneously.By comparing the retention time and MS/MS fragmentation of the two samples, the author can provide additional evidence that the AIP secreted by S. aureus is type I. Additionally, the author should provide a more detailed description of the identification procedure used in their study to ensure readers fully understand the process.2. The authors should clearly explain the differences and relationships between the bactericidal (Fig. 5c) and bacteriostatic (Fig. S7) effects observed in their study to help readers better understand the results.Additionally, the author should provide more detailed and clear information on "The bacterial inhibition test" to help readers understand the methodology used in the study.Minor comments: 3. It would be helpful for the authors to explain the reasoning behind their selection of the PldhL, P11, P32, and Pslp promoters.This would give readers a better understanding of why these specific promoters were chosen over others.
4. The authors should provide an explanation for their selection of the -35 and -10 regions of the P3 promoter as well as promoters P23 and P11.This would give readers more insight into why these specific regions and promoters were chosen for the study.
5. The authors should clarify whether the signal peptide N20 only boosts lysostaphin enzyme expression or if it also affects the quality of the enzyme.Additionally, the author should perform further tests to confirm the exact impact of N20 on lysostaphin enzyme expression and quality.
6.The authors should ensure that the description in Table 1 is centered and that there are no alignment issues in the table.Additionally, the author should provide clearer images of Fig. 2 and increase the fluorescence in Fig, 4 C to make it more visible.
Reviewer #2 (Comments for the Author): The paper of Haoran Li et al. described the creation of an engineered probiotic strain, Lactobacillus plantarum WCSF I for monitoring the treatment of S. aureus associated infections.The authors constructed an AgrQS system in L. plantarum capable of inhibiting S. aureus when detecting exogenous AIP nanomolar concentrations.According to my opinion, although the topic is interesting it is necessary to clarify some aspects and add some experiments to improve the paper as well as the understanding by the reader.
Major comments: 1.In the abstract the authors wrote that the engineering probiotics will be useful for monitoring the intestinal pathogens.S. aureus is an opportunistic pathogen associated with a wide range of infections, therefore, why do not set up an engineered probiotic strain particularly versus Enterobacteriaceae (like E. coli, Salmonella) or Listeria etc? Please specify your choice.2. Did the authors evaluate if the constructed could be subjected to the horizontal gene transfer?Please add some comments regarding this aspect.3. Did the authors evaluate a possible effect against other Staphylococcus species belonging to the microbiota?Please, test other Staphyloccocus species to demonstrate the selectivity.4. Did the authors evaluate a possible anti-biofilm activity of the supernatant produced by the L. plantarum modified strain?Please, demonstrate the quorum sensing inhibition. 5. Regarding the bacterial inhibition test (lines 295-305): the methods is just a qualitative method.Is there a method to quantify the component associated with the inhibition (lysostaphin)? 6.In literature a similar approach has been used in L. reuteri.Please, add comments regarding the paper "Programming probiotic L. reuteri as biosensor for S. aureus derived AIP 1 detection" by Lubkowicz et al. (2018).
7. Did the authors have any data regarding the safety of the modified strain on intestinal cell lines?8.An more thorough treatment of the materials and methods would be appreciated for a better understanding by the reader.The section material and methods is very confused.
Minor comments: 1. Please, specify the number of CFU/ml corresponding to the OD concentration in the section material and methods.2. Please write in italics the bacterial genus and species in the text and in the references.3.In the discussion the authors should explain the possible clinical applications deriving from the use of the modified strain.4. The quality of the images must be improved.The Figures are too much small and the quality is unfortunately bad. 5. Please add the number of independent experiments performed for the sections bacterial inhibition test and S. aureus supernatant analysis.

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