A 4-Hydroxybenzoic Acid-Mediated Signaling System Controls the Physiology and Virulence of Shigella sonnei

ABSTRACT Many bacteria use small molecules, such as quorum sensing (QS) signals, to perform intraspecies signaling and interspecies or interkingdom communication. Previous studies demonstrated that some bacteria regulate their physiology and pathogenicity by employing 4-hydroxybenzoic acid (4-HBA). Here, we report that 4-HBA controls biological functions, virulence, and anthranilic acid production in Shigella sonnei. The biosynthesis of 4-HBA is performed by UbiC (SSON_4219), which is a chorismate pyruvate-lyase that catalyzes the conversion of chorismate to 4-HBA. Deletion of ubiC caused S. sonnei to exhibit impaired phenotypes, including impaired biofilm formation, extracellular polysaccharide (EPS) production, and virulence. In addition, we found that 4-HBA controls the physiology and virulence of S. sonnei through the response regulator AaeR (SSON_3385), which contains a helix-turn-helix (HTH) domain and a LysR substrate-binding (LysR_substrate) domain. The same biological functions are controlled by AaeR and the 4-HBA signal, and 4-HBA-deficient mutant phenotypes were rescued by in trans expression of AaeR. We found that 4-HBA binds to AaeR and then enhances the binding of AaeR to the promoter DNA regions in target genes. Moreover, we revealed that 4-HBA from S. sonnei reduces the competitive fitness of Candida albicans by interfering with morphological transition. Together, our results suggested that the 4-HBA signaling system plays crucial roles in bacterial physiology and interkingdom communication. IMPORTANCE Shigella sonnei is an important pathogen in human intestines. Following previous findings that some bacteria employ 4-HBA as a QS signal to regulate biological functions, we demonstrate that 4-HBA controls the physiology and virulence of S. sonnei. This study is significant because it identifies both the signal synthase UbiC and receptor AaeR and unveils the signaling pathway of 4-HBA in S. sonnei. In addition, this study also supports the important role of 4-HBA in microbial cross talk, as 4-HBA strongly inhibits hyphal formation by Candida albicans. Together, our findings describe the dual roles of 4-HBA in both intraspecies signaling and interkingdom communication.

Major concerns: 1. Besides its potential role as a QS signal, 4-HBA has been long known as the precursor of Q8, which is an essential element for aerobic respiratory growth. Deletion of UbiC should completely eliminate Q8 biosynthesis and have wide metabolic consequences (attenuated virulence for example) and this could be an alternative way to explain the current data and should be extrapolated in the discussion. 2. The expression of UbiC is auto-regulated by 4-HBA, but 4-HBA does not activate AaeR nor AaeR directly regulates UbiC. In this sense, 4-HBA and AaeR are not analogous to the classical QS signal and receptor. The authors may comment on this point. 3. I'm surprised the RNA-seq data showed 4-HBA decreased the expression of ubiD, which was later showed to be potentially positively regulated by 4-HBA (EMSA data). Minor points: 1. Line 230: should be "4-HBA is a signaling ligand of AaeR. " 2. Line 316: should be "...to the promoter of ubiC (Fig. S11a, b), the expression of which was controlled by 4-HBA" Reviewer #2 (Comments for the Author): In the manuscript of Wang et al. " A 4-HBA mediated signaling system controls the physiology and virulence of Shigella sonnei", the authors found that extract of S. sonnei culture inhibits biofilm formation and development of C. albicans. The revealed that the active chemical is 4-HBA. In addition, the also found that 4-HBA binds a transcription regulator AaeR to enhance the activity of this TF in controlling downstream expression. This work is well designed and the experimental data is enough to support the conclusion, the results are interesting in understanding the bacteria-fungi interaction and interkingdom communication. I have several minor concerns on the work: 1. The authors found the mixture of S. sonnei extract inhibits biofilm formation and development of C. albicans ( Fig. 1) and revealed that 4-HBA is a major bioactive compound (Fig. S2). However, did 4-HBA the only active component in inhibition or there are other unidentified chemicals in the extract? How many percentages of 4-HBA in the mixture? 2. Lines 142-148, the authors checked the transcription level of MAPK marker genes and conclude the change of MAPK gene expressions is the reason to result in biofilm formation of fungi, this is not comprehensive because they did not check the other pathways. 3. Fig. 6d, in the EMSA experiment, there is only a 50 times competition, please added more competitions with various concentrations of un-labelled probe. In addition, please quantify the density of the bands Staff Comments:

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Point-to-point response to reviewers' suggestions for Spectrum04835-22
Reviewer comments: Reviewer #1 (Comments for the Author): This work demonstrated that 4-HBA act as a quorum sensing signaling molecule in Shigella sonnei by presenting the following evidence: 1. the production of 4-HBA in Shigella sonnei is positively correlated with the bacterial cell density; 2. the UbiC enzyme is responsible for synthesizing 4-HBA and expression of UbiC is auto-regulated by 4-HBA; 3. 4-HBA is recognized by the transcription regulator AaeR, enhances AaeR-DNA interaction, and influences the expression of a wide range of genes. Moreover, the authors also showed 4-HBA is a potential inter-kingdom signaling molecule interfering with morphological transition of Candida albicans. Although 4-HBA as a QS signal has been investigated in several bacterial stains, this work highlighted the dual roles of 4-HBA in both intraspecies signaling and interkingdom communication. Overall, this work is interesting and technically sound. Major concerns: 1. Besides its potential role as a QS signal, 4-HBA has been long known as the precursor of Q8, which is an essential element for aerobic respiratory growth. Deletion of UbiC should completely eliminate Q8 biosynthesis and have wide metabolic consequences (attenuated virulence for example) and this could be an alternative way to explain the current data and should be extrapolated in the discussion. Response: Thanks for your good suggestion, we have added this experiment result as Supplementary Figure 11 as suggested. Our result showed that the deletion of ubiC completely abolished the production of CoQ8 (Line 293-297).