Avishag Yehuda, Einav Malach, City Vanunu Ofri https://orcid.org/0009-0009-7162-2417, Leyla Slamti https://orcid.org/0000-0003-0275-8336, Shanny Hsuan Kuo https://orcid.org/0000-0001-8924-6960, Jonathan Z. Lau, Myung Whan Oh, John Adeoye, net Shlezinger, Didier Lereclus https://orcid.org/0000-0001-9047-9104, Gee W. Lau https://orcid.org/0000-0002-7962-3950and Zvi Hayouka https://orcid.org/0000-0003-3582-4029 [email protected]Authors Info & Affiliations
Edited by E. Greenberg, University of Washington, Seattle, WA; received January 19, 2023; accepted June 23, 2023
August 22, 2023
120 (35) e2301045120
Significance
Here, we report how interception of quorum sensing (QS) communication in Bacillus cereus (Bc) as a model pathogen obliterates the ability to control the host immune system. We have designed a peptide-based QS inhibitor that suppresses Bc virulence factor expression and attenuates infectivity in mouse models. We have demonstrated that the QS peptidic inhibitor blocks host immune system–mediated eradication by reducing the expression and secretion of major Bc toxins. Our findings show that Bc infectivity is regulated by the QS circuit that mediates destruction of the host immunity, thus revealing a new strategy to limit Bc virulence and enhance host defense.
Abstract
Subverting the host immune system is a major task for any given pathogen to assure its survival and proliferation. For the opportunistic human pathogen Bacillus cereus (Bc), immune evasion enables the establishment of potent infections. In various species of the Bc group, the pleiotropic regulator PlcR and its cognate cell–cell signaling peptide PapR7 regulate virulence gene expression in response to fluctuations in population density, i.e., a quorum-sensing (QS) system. However, how QS exerts its effects during infections and whether PlcR confers the immune evading ability remain unclear. Herein, we report how interception of the QS communication in Bc obliterates the ability to affect the host immune system. Here, we designed a peptide-based QS inhibitor that suppresses PlcR-dependent virulence factor expression and attenuates Bc infectivity in mouse models. We demonstrate that the QS peptidic inhibitor blocks host immune system–mediated eradication by reducing the expression of PlcR-regulated major toxins similarly to the profile that was observed for isogenic strains. Our findings provide evidence that Bc infectivity is regulated by QS circuit-mediated destruction of host immunity, thus reveal a interesting strategy to limit Bc virulence and enhance host defense. This peptidic quorum-quenching agent constitutes a readily accessible chemical tool for studying how other pathogen QS systems modulate host immunity and forms a basis for development of anti-infective therapeutics.
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Data, Materials, and Software Availability
All study data are included in the article and/or SI Appendix.
Acknowledgments
The authors would like to Eugénie Huillet and Ludovic Bridoux for the construction of the B. cereus ATCC 14579 papR mutant strains.
Author contributions
A.Y., N.S., D.L., and Z.H. designed research; A.Y., E.M., S.V.O., L.S., S.H.K., J.Z.L., M.W.O., J.A., G.W.L., and Z.H. performed research; A.Y. and L.S. contributed new reagents/analytic tools; A.Y. and E.M. analyzed data; and A.Y. and Z.H. wrote the paper.
Competing interests
The authors declare no competing interest.
Supporting Information
References
1
G. M. Dunny, B. A. Leonard, Cell-cell communication in gram-positive bacteria. Year. Rev. Microbiol. 51527–64 (1997).
2
M. B. Miller, B. L. Bassler, Quorum sensing in bacteria. Year. Rev. Microbiol. 55165–199 (2001).
3
B. L. Bassler, R. Losick, Bacterially speaking. Cell 125237–246 (2006).
4
S. T. Rutherford, B. L. Bassler, Bacterial quorum sensing: Its role in virulence and possibilities for its control. Cold Spring Harb. Perspec. Med. 2a012427 (2012).
5
L. Slamti, S. Perchat, E. Huillet, D. Lereclus, Quorum sensing in Bacillus thuringiensis is required for completion of a full infectious cycle in the insect. Toxins 62239–2255 (2014).
6
A. Rajput, K. Kaur, M. Kumar, SigMol: Repertoire of quorum sensing signaling molecules in prokaryotes. Nucleic Acids Res. 44D634–D639 (2016), https://doi.org/10.1093/nar/gkv1076.
7
K. Papenfort, B. L. Bassler, Quorum sensing signal-response systems in Gram-negative bacteria. Nat. Rev. Microbiol. 14576–588 (2016).
8
M. B. Neiditch, G. C. Capodagli, G. Prehna, M. J. Federle, Genetic and structural analyses of RRNPP intercellular peptide signaling of Gram-positive bacteria. Annu. Rev. Genet. 51311–333 (2017).
9
C. Grandclément, M. Tannières, S. Moréra, Y. Dessaux, D. Faure, Quorum quenching: Role in nature and applied developments. FEMS Microbiol. Rev. 4086–116 (2016), https://doi.org/10.1093/femsre/fuv038.
10
S. Uroz, Y. Dessaux, P. Oger, Quorum sensing and quorum quenching: The Yin and Yang of bacterial communication. ChemBioChem 10205–216 (2009).
11
P. Piewngam, J. Chiou, P. Chatterjee, M. Otto, Alternative approaches to treat bacterial infections: Targeting quorum-sensing. Expert Rev. Anti Infect. Ther. 18499–510 (2020).
12
M. A. Welsh, H. E. Blackwell, Chemical probes of quorum sensing: From compound development to biological discoverya. FEMS Microbiol. Rev. 40774–794 (2016).
13
R. C. Allen, R. Popat, S. P. Diggle, S. P. Brown, Targeting virulence: Can we make evolution-proof drugs? Nat. Rev. Microbiol. 12300–308 (2014).
14
D. A. Rasko, V. Sperandio, Anti-virulence strategies to combat bacteria-mediated disease. Nat. Rev. Drug Discov. 9117–128 (2010).
15
C. Walsh, Molecular mechanisms that confer antibacterial drug resistance. Nature 406775–781 (2000).
16
W. R. J. D. Galloway, J. T. Hodgkinson, S. Bowden, M. Welch, D. R. Spring, Applications of small molecule activators and inhibitors of quorum sensing in Gram-negative bacteria. Trends Microbiol. 20449–458 (2012).
17
R. J. Case, M. Labbate, S. Kjelleberg, AHL-driven quorum-sensing circuits: Their frequency and function among the Proteobacteria. ISME J. 2345–349 (2008).
18
M. E. Mattmann, H. E. Blackwell, Small molecules that modulate quorum sensing and control virulence in pseudomonas aeruginosa. J. Org. Chem. 756737–6746 (2010).
19
P. D. Utari, R. Setroikromo, B. N. Melgert, W. J. Quax, PvdQ quorum quenching acylase attenuates Pseudomonas aeruginosa virulence in a mouse model of pulmonary infection. Front. Cell. Infect. Microbiol. 8119 (2018).
20
D. N. McBrayer, C. D. Cameron, Y. Tal-Gan, Development and utilization of peptide-based quorum sensing modulators in Gram-positive bacteria. Org. Biomol. Chem. 187273–7290 (2020).
21
Y. Tal-Gan, M. Ivancic, G. Cornilescu, C. C. Cornilescu, H. E. Blackwell, Structural characterization of native autoinducing peptides and abiotic analogues reveals key features essential for activation and inhibition of an agrc quorum sensing receptor in staphylococcus aureus. J. Am. Chem. Soc. 13518436–18444 (2013).
22
Y. Tal-Gan, D. M. Stacy, M. K. Foegen, D. W. Koenig, H. E. Blackwell, Highly potent inhibitors of quorum sensing in staphylococcus aureus revealed through a systematic synthetic study of the group-III autoinducing peptide. J. Am. Chem. Soc. 1357869–7882 (2013).
23
G. Lina et al., Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus. Mol. Microbiol. 28655–662 (1998).
24
G. J. Lyon, P. Mayville, T. W. Muir, R. P. Novick, Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC. Proc. Natl. Acad. Sci. U.S.A. 9713330–13335 (2000).
25
E. A. George, R. P. Novick, T. W. Muir, Cyclic peptide inhibitors of staphylococcal virulence prepared by Fmoc-based thiolactone peptide synthesis. J. Am. Chem. Soc. 1304914–4924 (2008).
26
L. Zhu, G. W. Lau, Inhibition of competence development, horizontal gene transfer and virulence in streptococcus pneumoniae by a modified competence stimulating peptide. PLoS Pathog. 7e1002241 (2011).
27
Y. Yang et al., Designing cyclic competence-stimulating peptide (CSP) analogs with pan-group quorum-sensing inhibition activity in Streptococcus pneumoniae. Proc. Natl. Acad. Sci. U.S.A. 1171689–1699 (2020).
28
J. Nakayama et al., Development of a peptide antagonist against fsr quorum sensing of Enterococcus faecalis. ACS Chem. Biol. 8804–811 (2013).
29
D. N. McBrayer, C. D. Cameron, B. K. Gantman, Y. Tal-Gan, Rational design of potent activators and inhibitors of the Enterococcus faecalis Fsr quorum sensing circuit. ACS Chem. Biol. 132673–2681 (2018).
30
D. N. McBrayer, B. K. Gantman, C. D. Cameron, Y. Tal-Gan, An Entirely solid phase peptide synthesis-based strategy for synthesis of gelatinase biosynthesis-activating pheromone (GBAP) analogue libraries: Investigating the structure-activity relationships of the Enterococcus faecalis quorum sensing signal. Org. Lett. 193295–3298 (2017).
32
A. Yehuda, L. Slamti, E. Malach, D. Lereclus, Z. Hayouka, Elucidating the hot spot residues of quorum sensing peptidic autoinducer PapR by multiple amino acid replacements. Front. Microbiol. 101246 (2019), https://doi.org/10.3389/fmicb.2019.01246.
33
L. P. Stenfors Arnesen, A. Fagerlund, P. E. Granum, From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Rev. 32579–606 (2008).
35
E. Helgason et al., Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis–one species on the basis of genetic evidence. Appl. Envi ron. Microbiol. 662627–2630 (2000).
36
P. E. Granum, T. Lund, Bacillus cereus and its food poisoning toxins. FEMS Microbiol. Lett. 157223–228 (1997).
37
E. J. Bottone, Bacillus cereus, a volatile human pathogen. Clin. Microbiol. Rev. 23382–398 (2010).
38
B. Glasset et al., Bacillus cereus, a serious cause of nosocomial infections: Epidemiologic and genetic survey. PLoS One 131–19 (2018).
39
D. Enosi Tuipulotu, A. Mathur, C. Ngo, S. M. Man, Bacillus cereus: Epidemiology, virulence factors, and host-pathogen interactions. Trends Microbiol. 29458–471 (2021).
40
E. Hernandez, F. Ramisse, J. P. Ducoureau, T. Cruel, J. D. Cavallo, Bacillus thuringiensis subsp. konkukian (Serotype H34) superinfection: Case report and experimental evidence of pathogenicity in immunosuppressed mice. J. Clin. Microbiol. 362138–2139 (1998).
41
N. Gilois et al., Growth-related variations in the Bacillus cereus secretome. Proteomics 71719–1728 (2007).
42
S. L. Tran et al., Haemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages. Cell Microbiol. 1392–108 (2011).
44
M. Gohar et al., The PlcR virulence regulon of Bacillus cereus. PLoS One 3e2793 (2008).
46
S. Salamitou et al., The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects. Microbiology 1462825–2832 (2000).
47
D. J. Beecher, T. W. Olsen, E. B. Somers, A. C. L. Wong, Evidence for contribution of tripartite hemolysin BL, phosphatidylcholine-preferring phospholipase C, and collagenase to virulence of Bacillus cereus endophthalmitis. Infect. Immunity 685269–5276 (2000).
48
D. J. Beecher, A. C. L. Wong, Cooperative, synergistic and antagonistic haemolytic interactions between haemolysin BL, phosphatidylcholine phospholipase C and sphingomyelinase from Bacillus cereus. Microbiology 1463033–3039 (2000).
49
A. P. Pomerantsev, K. V. Kalnin, M. Osorio, S. H. Leppla, Phosphatidylcholine-specific phospholipase C and sphingomyelinase activities in bacteria of the Bacillus cereus group. Infect. Immunity 716591–6606 (2003).
50
I. Sastalla et al., The Bacillus cereus Hbl and Nhe tripartite enterotoxin components assemble sequentially on the surface of target cells and are not interchangeable. PLoS One 8e76955 (2013).
51
J. Liu et al., Sequential CRISPR-based screens identify LITAF and CDIP1 as the Bacillus cereus hemolysin BL toxin host receptors. Cell Host Microbe 28402–410.e5 (2020).
52
L. Slamti, D. Lereclus, A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group. EMBO J. 214550–4559 (2002).
53
M. Gominet, L. Slamti, N. Gilois, M. Rose, D. Lereclus, Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence. Mol. Microbiol. 40963–975 (2001).
54
R. Grenha et al., Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR. Proc. Natl. Acad. Sci. U.S.A. 1101047–1052 (2013).
55
D. Lereclus, H. Agaisse, M. Gominet, S. Salamitou, V. Sanchis, Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase. J. Bacteriol. 1782749–2756 (1996).
56
H. Agaisse, M. Gominet, O. A. Okstad, A. B. Kolstø, D. Lereclus, PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis. Mol. Microbiol. 321043–1053 (1999).
57
M. Gohar et al., Two-dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon. Proteomics 2784–791 (2002).
59
L. Bouillaut et al., Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides. Nucleic Acids Res. 363791–3801 (2008).
60
D. Lereclus, O. Arantès, J. Chaufaux, M. Lecadet, Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol. Lett. 51211–217 (1989).
61
Y. Yang et al., Structure-activity relationships of the competence stimulating peptides (CSPs) in Streptococcus pneumoniae Reveal motifs critical for intra-group and cross-group ComD receptor activation. ACS Chem. Biol. 121141–1151 (2017), https://doi.org/10.1021/acschembio.7b00007.
62
C. J. McGill, R. J. Lu, B. A. Benayoun, Protocol for analysis of mouse neutrophil NETosis by flow cytometry. STAR Protoc. 2100948 (2021).
63
M. Swamydas, M. S. Lionakis, Isolation, purification and labeling of mouse bone marrow neutrophils for functional studies and adoptive transfer experiments. J. Vis. Exp.e50586 (2013).
64
R. A. Scheltema et al., The Q exactive HF, a benchtop mass spectrometer with a pre-filter, high-performance quadrupole and an ultra-high-field orbitrap analyzer. Mol. Cell. Proteomics 133698–3708 (2014).
65
J. Cox et al., Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics 132513–2526 (2014).
67
R. Visiello, S. Colombo, E. Carretto, “Chapter 3–Bacillus cereus Hemolysins and Other Virulence Factors,” in The Diverse Faces of Bacillus cereus (Academic Press, 2016), pp. 35–44.
68
N. Ramarao, D. Lereclus, The InhA 1 metalloprotease allows spores of the B. cereus group to escape macrophages. Cell Microbiol. 71357–1364 (2005).
69
S. L. Tran, E. Guillemet, M. Gohar, D. Lereclus, N. Ramarao, CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation, and virulence. J. Bacteriol. 1922638–2642 (2010).
70
B. Amulic, C. Cazalet, G. L. Hayes, K. D. Metzler, A. Zychlinsky, Neutrophil function: From mechanisms to disease. Year. Rev. Immunol. 30459–489 (2012).
71
E. T. Livingston, M. H. Mursalin, M. C. Callegan, A pyrrhic victory: The PMN response to ocular bacterial infections. Microorganisms 7537 (2019).
72
M. Garcia Chavez et al., Synthesis of fusidic acid derivatives yields a potent antibiotic with an improved resistance profile. ACS Infect. Dis. 7493–505 (2021).
73
S. E. Cheah et al., New pharmacokinetic/pharmacodynamic studies of systemically administered colistin against Pseudomonas aeruginosa and Acinetobacter baumannii in mouse thigh and lung infection models: Smaller response in lung infection. J. Antimicrob. Chemother. 703291–3297 (2015).
74
M. S. Gilmore, A. L. Cruz-Rodz, M. Leimeister-Wachter, J. Kreft, W. Goebel, A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: Nucleotide sequence and genetic linkage. J. Bacteriol. 171744–753 (1989).
75
M. G. Netea, J. Quintin, J. W. M. Van Der Meer, Trained immunity: A memory for innate host defense. Cell Host Microbe 9355–361 (2011).
76
H. K. Lehman, B. H. Segal, The role of neutrophils in host defense and disease. J. Allergy Clin. Immunol. 1451535–1544 (2020).
77
S. H. E. Kaufmann, A. Dorhoi, Molecular determinants in phagocyte-bacteria interactions. Immunity 44476–491 (2016).
78
C. Leseigneur, P. Lê-Bury, J. Pizarro-Cerdá, O. Dussurget, Emerging evasion mechanisms of macrophage defenses by pathogenic bacteria. Front. Cell Infect. Microbiol. 101–9 (2020).
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Published in
Proceedings of the National Academy of Sciences
Vol. 120 | No. 35
August 29, 2023
Classifications
Copyright
Data, Materials, and Software Availability
All study data are included in the article and/or SI Appendix.
Submission history
Received: January 19, 2023
Accepted: June 23, 2023
Published online: August 22, 2023
Published in issue: August 29, 2023
Keywords
- quorum sensing
- quorum quenching
- host–pathogen interaction
- neutrophils
- peptides
Acknowledgments
The authors would like to Eugénie Huillet and Ludovic Bridoux for the construction of the B. cereus ATCC 14579 papR mutant strains.
Author Contributions
A.Y., N.S., D.L., and Z.H. designed research; A.Y., E.M., S.V.O., L.S., S.H.K., J.Z.L., M.W.O., J.A., G.W.L., and Z.H. performed research; A.Y. and L.S. contributed new reagents/analytic tools; A.Y. and E.M. analyzed data; and A.Y. and Z.H. wrote the paper.
Competing Interests
The authors declare no competing interest.
Notes
This article is a PNAS Direct Submission.
Authors
Affiliations
Avishag Yehuda
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agricultural, Food & Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel
Einav Malach
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agricultural, Food & Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agricultural, Food & Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel
Micalis Unit, Domaine de La Minière, Mixed Research Unit 1319, National Institute for Agronomic Research, 78280 Guyancourt, France
Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802
Jonathan Z. Lau
Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802
Myung Whan Oh
Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802
John Adeoye
Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agricultural, Food & Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel
net Shlezinger
Koret School of Veterinary Medicine, The Robert H. Smith Faculty of Agricultural, Food & Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel
Micalis Unit, Domaine de La Minière, Mixed Research Unit 1319, National Institute for Agronomic Research, 78280 Guyancourt, France
Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, IL 61802
Institute of Biochemistry, Food Science and Nutrition, The Robert H. Smith Faculty of Agricultural, Food & Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel
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