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1. Staphylococcus aureus
2. Two-Component Signaling System (TCS)
3. Staphylococcus aureus virulence regulating mechanism: AgrBDCA
4. Lactobacillus plantarum WCFS1 quorum sensing system: PlnABCD
5. RNAIII inhibiting peptide (RIP) of Staphylococcus aureus

1. Staphylococcus aureus

Staphylococcus aureus is a facultatively anaerobic, gram-positive coccus which forms large round yellow colonies when grown on agar plate [1]. It was reported that approximately 20% of human population are long-term carriers of at least one S. aureus strain [2]. As a versatile and potential dangerous pathogen, S. aureus can cause a wide range of diseases varying from skin flora infections to invasive infections such as sepsis syndrome [1, 3]. Many of these diseases are associated with regulation factors involved in toxin synthesis in S. aureus. These bacterial regulation factors play critical roles in virulence production; the strains that are defective in producing toxin regulation factors always show weakened virulence [4]. The aforementioned information suggests a novel therapy, which involves the inhibition of toxin regulation factors, for diseases caused by S. aureus.

2. Localization Test of Chimeric AIP Receptor

In many bacterial species, Two-Component Signaling System (TCS) plays a prominent role in quorum sensing. A classical TCS consists of a transmembrane receptor, which is a histidine protein kinase (HPK), and a cytoplasmic response regulator (RR) [5]. When bound by autoinducing peptides (AIP), the transmembrane receptor HPK passes a phosphate group to the downstream cytoplasmic response regulator RR. The cytoplasmic response regulator will then up-regulates or down-regulates various virulence and bacteriocin productions under specific situations [6]. Figure 1 below illustrates the components and transduction pathways of a typical two-component signaling system:

3. Staphylococcus aureus virulence regulating mechanism: AgrBDCA

A chromosomal locus agr system in S. aureus consists of two transcription units RNA II and RNA III; they are responsible for self-regulation and effect response respectively [7, 8]. RNA II, driven by P2 promoter, is a polycistronic mRNA containing four open reading frames: agrB, agrD, agrC and agrA, and are responsible for agr quorum sensing in S. aureus [9]. AgrB is an integral membrane protein; AgrD is a pro-peptide secreted with the aid of AgrB, yielding an octapeptide pheromone autoinducing peptide (AIP); AgrC and AgrA compose a bacterial two-component signal transduction system, in which AgrC, a membrane associated protein, serves as a receptor and AgrA acts as a DNA-binding response regulator[10,11, 12]. Once the transmembrane receptor domain of AgrC is bound by AIP, its cytoplasmic HPK domain will pass a phosphate group downwards to AgrA, the response regulator. AgrA functions as a transcription factor and activates the two promoters P2 and P3 [13, 14]. RNA III, driven by P3 promoter, acts as a regulatory factor to activate numerous toxin production genes and encode for β-hemolysin [15]. Figure 2 below shows the pathways involved in S. aureus AgrBDCA system:

It has been reported that there are altogether 4 types of agr loci; each type of agr locus encodes AgrC and corresponding AIP slightly different from others. AgrC is only activated by AIP belonging to the same group and is usually inhibited by AIP from other groups [16]. Different groups of AgrC and their corresponding AIPs are highly involved in various human diseases or infections. For example, food poisoning caused by staphylococcal enterotoxins is usually associated with agr group I and agr group II S. aureus; toxic shock syndrome toxin 1 (TSST-1), which results in high fever, low blood pressure and malaise of human, is produced by agr group III S. aureus [17].

The synthesis of RNAIII is regulated by two Staphylococcal quorum sensing systems SQS 1 and SQS 2. SQS 1 consists of a 277-amino-acid RNAIII-activating protein (RAP) and a 167-amino-acid target of RNAIII-activating protein (TRAP) which is membrane associated [15]; SQS 2 consists of molecules encoded by agr system [18]. As S. aureus multiplies, RAP is secreted. Once RAP reaches a threshold concentration, it will induce the histidine phosphorylation of TRAP, which leads to the activation of agr system and therefore induces the production of RNAII during the mid-exponential growth phase [15]. After the activation of agr system, phosphorylation of AgrC is induced by secreted AIP, followed by the production of RNAIII [18].

The characteristics of S. aureus AgrBDCA system aforementioned make it possible to build an engineered AIP sensor on the membrane of a non-pathogenic bacterial species. The engineered AIP sensor, localized on the non-virulent bacteria, could therefore detect the presence of AIP molecules released by S. aureus. Upon receiving this signal, the non-virulent bacteria could be automatically induced to synthesize AIP’s competitor, RNAIII inhibiting peptide (RIP), and hence, repress the activation of AgrC and toxin production in S. aureus.

4. Lactobacillus plantarum WCFS1 quorum sensing system: PlnABCD

Several agr-like quorum sensing systems in Lactobacillus plantarum WCFS1 have been identified, among which the PlnABCD system is the best studied [5]. The PlnABCD system, similar to the AgrBDCA system, is a self-regulating system in L. plantarum WCFS1. The transmembrane receptor HPK PlnB, together with two response regulators PlnC and PlnD, constitutes a two-component signaling system (TCS) in L. plantarum WCFS1. PlnA, the inducing peptide (IP) of L. plantarum WCFS1, activates PlnB and thereby phosphoylates PlnC or PlnD. PlnC and PlnD are upregulator and downreuglater of plnA promoter respectively, and therefore, would activate/suppress the transcription initiation of plnABCD. The transcription of plnABCD is usually associated with Lactobacillus plantarum WCFS1 bacteriocin production [19]. Figure 3 below shows the pathways involved in Lactobacillus plantarum WCFS1 PlnABCD system:

Transmembrane signal sensors of both PlnABCD and AgrBDCA systems, i.e. PlnB (of L. plantarum WCFS1) and AgrC (of S. aureus), belong to a same subgroup of histidine protein kinases – the HPK10 subfamily. PlnB and AgrC share a highly homologous cytoplasmic HPK domain regarding amino acid sequence, tertiary structure and biochemical function [14, 19]. However, the sequence of PlnB and AgrC’s transmembrane domain are distinct from each other, probably due to the ligand binding specificity required by their corresponding signaling molecules.

Based on the homology between AgrBDCA and PlnABCD at the cytoplasmic HPK domain, we decided to construct a chimeric AIP sensor in Lactobacillus. Such a fusion receptor would link the transmembrane signal sensing domain of AgrCand the cytoplasmic HPK domain of PlnB. By localizing such a fusion protein on Lactobacillus plasma membrane, it is hoped that Lactobacillus can successfully detect the presence of autoinducing peptides (AIPs) produced by S. aureus. Upon the detection of AIP molecules, the chimeric protein kinase will transduce the signal to L. plantarum WCFS1intrinsic downstream pathways.

5. RNAIII inhibiting peptide (RIP) of Staphylococcus aureus

The S. aureus RN833 is a mutant strain of Foggi (now listed as RN831) as it no longer produces α-hemolysin. Instead, RNAIII inhibiting peptide, an inhibitor of agr system abbreviated as RIP, was discovered and tested in 1993 [20]. Heptapeptide RIP has been proved to be an effective inhibitor to reduce virulence and prevent infections of S. aureus [18, 21].

The sequence of RIP was identified as YSPXTNF, where X can be a Cys, a Trp, or a modified amino acid [18]. It is similar to the NH2 terminal sequence of RAP IKKYKPITN [22], the natural ligand of TRAP receptor. Therefore, it is hypothesized that RAP and RIP bind to the same receptor, TRAP [23]. Through competing with RAP on binding to TRAP and thus interfering with the activation of agr system, RIP inhibits the expression of RNAIII and the toxin genes in S. aureus. Based on the fact that TRAP is highly conserved among S. aureus strains, RIP is supposed to inhibit pathogenesis of most S. aureus strains infections [18].

A synthetic RIP derivative YKPWTNF was tested for its ability to inhibit RNAIII synthesis in vitro and to prevent infection in vivo [23]. The natural RIP and its synthetic derivatives have higher binding affinity to TRAP than RAP, and therefore would inhibit the normal pathway of TRAP by induced by RAP [24]. Researches indicated that RIP YKPWTNF can suppress virulence of any S. aureus strain so far tested [18, 23].

A 13-residue dermaseptin derivative DD13, identified as ALWKTLLKKVLKA, is believed to have the ability to kill bacteria via membrane disruption. The synthetic hybrid construct DD13-RIP derivative ALWKTLLKKVLKAYSPWTNF-CONH2 has been tested for the ability to suppress quorum sensing of S. aureus in vitro and shows synergistic effect comparing with single synthetic RIP derivative YKPWTNF-CONH2 [25]. Hence, by functionally expressing the hybrid peptide DD13-RIP derivative, S. aureus infections may be inhibited through restraining quorum sensing systems. However, since the hybrid construct is made on a DNA level, after transcription and translation, the synthetic RIP will be in the form of ALWKTLLKKVLKAYSPWTNF-COOH other than the synthetic one ALWKTLLKKVLKAYSPWTNF-CONH2, which may lead to an altered efficiency.

References for Background

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  2. Kluytmans, J., van Belkum, A., & Verbrugh, H. (1997). Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms, and associated risks. Clin Microbiol Rev, 10(3), 505-520. 

  3. Franklin, D., & Lowy, F.D. (1998). Staphylococcus aureus infections. N Engl J Med339(27), 520-532. 

  4. Kiran, M.D., Adikesavan, N.V., Cirioni, O., Giacometti, A., Silvestri, C., Scalise, G., Ghiselli, R., Saba, V., Orlando, F., Shoham, M., & Balaban, N. (2008). Discovery of a quorum-sensing inhibitor of drug-resistant Staphylococcal infections by structure-based virtual screening. Mol Pharmacol, 73(5), 718-726. 

  5. Sturme, M.H.J., Francke, C., Siezen, R.J., de Vos, W.M., & Kleerebezem, M. (2007). Making sense of quorum sensing in lactobacilli: a special focus on lactobacillus plantarum wcfs1Microbiology, 153, 3939–3947.  

  6. Kleerebezem, M., Quadri, L. E. N., Kuipers, O. P. & De Vos, W. M.(1997). Quorum sensing by peptide pheromones and two-componentsignal-transduction systems in Gram-positive bacteria. Molecular Microbiology, l24, 895–904.  

  7. Morfeldt, E., Janzon, L., Arvidson, S., and Lofdahl, S. (1988) Cloning of a chromosomal locus (exp) which regulates the expression of several exoprotein genes in Staphylococcus aureus. Mol Gen Genet, 211, 435–440.  

  8. Ji, G., Beavis, R.C., and Novick, R.P. (1995) Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc Natl Acad Sci USA, 92, 12055–12059.  

  9. Novick, R.P., Ross, H.F., Projan, S.J., Kornblum, J., Kreiswirth, B., & Moghazeh, S. (1993). Synthesis of Staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J12(10), 3967-3975.  

  10. Koenig, R.L., Ray, J.L., Maleki, S.J., Smeltzer, M.S., Hurlburt, B.K. (2004). Staphylococcus aureus AgrA binding to the RNAIII-agr regulatory region. J of Bacteriol, 186(22), 7549-7555.  

  11. Korem, M., Sheoran , A.S., Gov, Y., Tzipori, S., Borovok, I., & Balaban, N. (2003). Characterization of RAP, a quorum sensing activator of Staphylococcus aureusFEMS Microbiol Lett, 223(2), 167-175.  

  12. Balaban, N., Goldkorn, T., Gov, Y., Hirshberg, M., Koyfman, N., Matthews, H.R., Nhan, R.T., Singh, B., Uziel, O. (2001). Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating protein (TRAP). J Biol Chem276(4), 2658-2667. 

  13. Novick RP, Projan SJ, Kornblum J, Ross HF, Ji G, et al. 1995. The agr P2 operon: an autocatalytic sensory transduction system in taphylococcus aureus. Mol. Gen. Genet, 248, 446–58.   

  14. Novick, R. P. & Geisinger, E. (2008). Quorum sensing in Staphylococci. Annual Review of Genetics, 42, 541–64.  

  15. Gov, Y., Borovok, I., Korem, M., Singh, V.K., Jayaswal, R.K., Wilkinson, B.J., Rich, S.M., & Balaban, N. (2004). Quorum sensing in Staphylococci is regulated via phosphorylation of three conserved histidine residues. J Biol Chem, 279(15), 14665-14672.  

  16. Geisinger, E., George, E.A., Muir, T.W., & Novick, R.P.(2008). Identification of ligand specificity determinants in agrc, the staphylococcus aureus quorum-sensing receptor . The Journal of Biological Chemistry, 283(14), 8930–8938. 

  17. Jarraud, S., Mougel, C., Thioulouse, J., & Lina, G. (2002). Relationships between staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease . Infection and Immunity, 70(2), 631–641.  

  18. Gov, Y., Bitler, A., Dell'Acqua, G., Torres, J.V., & Balaban, N. (2001). RNAIII inhibiting peptide (RIP), a global inhibitor of Staphylococcus aureus pathogenesis: structure and function analysis. Peptides22(10), 1609-1620.  

  19. Johnsborg, O., Diep, D. B. & Nes, N. F. (2003). Structural analysis of the peptide pheromone receptor plnB, a histidine protein kinase from Lactobacillus plantarum. Journal of Bacteriology, 185 (23), 6913–6920.  

  20. Balaban, N., & Novick, R.P. (1995). Autocrine regulation of toxin synthesis by Staphylococcus aureusProc Natl Acad Sci U S A92(5), 1619-1623.   

  21. Costerton JW, Montanaro L, Arciola CR. (2007). Bacterial communications in implant infections: a target for an intelligence war. Int J Artif Organs. 30(9):757-63.

  22. Novick, R.P., Ross, H.F., Figueiredo, A.M.S., Abramochkin, G., Muir, T., Balaban, N., Singh, B., Goldkorn, T., Rasooly, A., Torres, J.V., & Uziel, O. (2000). Activation and inhibition of the Staphylococcal agr system. Science287, 391. 

  23. Balaban, N., Collins, L.V., Cullor, J.S., Hume, E.B., Medina-Acosta, E., Vieira da Motta, O., O'Callaghan, R., Rossitto, P.V., Shirtliff, M.E., Serafim da Silveira, L., Tarkowski, A., & Torres, J.V. (2000). Prevention of diseases caused by Staphylococcus aureus using the peptide RIP. Peptides21(9), 1301-1311. 

  24. Balaban, N., Goldkorn, T., Nhan, R.T., Dang, L.B., Scott, S., Ridgley, R.M., Rasooly, A., Wright, S.C., Larrick, J.W., Rasooly, R., & Carlson, J.R. (1998). Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus. Science280(5362), 438-440. 

  25. Balaban, N., Gov, Y., Giacometti, A., Cirioni, O., Ghiselli, R., Mocchegiani, F., Orlando, F., D'Amato, G., Saba, V., Scalise, G., Bernes, S., & Mor, A. (2004). A chimeric peptide composed of a dermaseptin derivative and an RNAIII-inhibiting peptide prevents graft-associated infections by antibiotic-resistant StaphylococciAntimicrob Agents Chemother48(7), 2544-2550.