MRSA: RIP?

Let us disarm a bit. Regarding my comment on Dr Mark Smeltzer article, I would like to set the record straight: By NO means I questioned (or even intended to question) the peer-review process for those manuscripts. That was never the issue. Perhaps, brevity compromised the meaning of my comment. What I did mean is that, regarding the mutagenesis results neither study directly addressed the function of TRAP. I did not question the mutagenesis data. I did question the comment or its interpretation made as being evidence against the function of TRAP. Let us keep in mind that TRAP was originally discovered by in vivo phosphorylation studies, where RAP upregulated and RIP downregulated its phosphorylation. Thus, for the better or worst, either paper case would have been stronger if phosphorylation studies and western blotting had been performed. The question underlining (clearly not intended to challenge the articles but raised with the intention to help the reader _ myself included_ understand what is going on in the field) can be summarized to whether the mutant and parental strains were checked for any difference in TRAP phosphorylation. (In vivo phosphorylation assays: Bacteria+P32+RAP=increase in TRAP phosphorylation versus Bacteria+P32+RIP=decrease in TRAP phosphorylation). Although some time ago I had a happy collaboration with Dr Balaban, I have no compromise with her success or failure. I write as an independent source. I made that clear in my previous comment when I asked about the agr mutant strains. If TRAP regulates DNA repair (as now revealed by Dr Balaban), then when TRAP is mutated (directly or indirectly) many nonsense mutations should be expected, making TRAP an even better target of intervention. I hope the next articles (peer-reviewed) from either side of the debate shed a brighter light on those questions.

Here is what we know:
1. RIP is extremely effective in suppressing infections, in concentrations similar to those commonly used in antibiotic treatment (tested in numerous settings)
2. TRAP and RAP are very effective as vaccine target sites (tested in mice and in cows).
3. TRAP was originally discovered by in vivo phosphorylation studies, where RAP upregulated and RIP downregulated its phosphorylation.
This was confirmed by in vitro studies, devoid of other cellular components: RIP inhibits and RAP activates TRAP phosphorylation.
4. RAP upregulates and RIP downregulates the agr (RNAII and RNAIII production).

Obvious conclusions from above:
RIP and RAP regulate the agr via TRAP. Perhaps we are wrong and they operate in parallel rather than in tandem?
Either way, MRSA: R.I.P. still holds.

Now to mutagenesis studies:
While studying analogous systems in other gram-positive bacteria, we discovered that TRAP and its ortholog regulate DNA repair and stress response mechanisms.
When TRAP or its ortholog are inactivated, there is accumulation of nonsense mutations in multiple regulators of virulence (like the agr), leading to complete inactivation of virulence genes. While this makes the study of these systems very difficult (and consequently controversial), it once again leads us to the same conclusions:
TRAP is a very good target site for therapy and thus RIP, which inhibits its phosphorylation, also scientifically makes a lot of sense.

Now to the politics and to human behavior:
Creating a contentious environment will not facilitate further understanding of the RAP/RIP/TRAP system, virulence or future therapeutics. Instead of suppressing publications, grant proposals, lectures etc, why not open the subject further? After all, the public funds us. We at least owe the public a proper due process of science.

As for specific remarks:
To Michael Otto: I really do not remember what you had suggested at a conference back in 2003. I had asked you before to send me your comments by email, but you refused. So once again I am asking: can you please email me a list of experiments you would have liked to see done? Perhaps we could work on that collaboratively.

To Mark Smeltzer and Laura Tsang: Please do read the 2008 Springer book. You may be pleasantly surprised. Until then-try to promote rather than suppress what may be of great value to public health.

We had hoped to avoid this ?sad debate? by letting our work rest on its merits, but it appears we will not be afforded that opportunity. Dr. Costerton refers to a ?profound sense of sadness concerning researchers who try to drag their colleagues down?. He goes on to suggest the ?moral? that we ?talk to each other decently and don't publish to discredit, because the field will clarify as more data emerges?. While he does not make clear exactly who or what he is referring to, phrasing these comments in this fashion clearly implies that his concerns include our paper (Tsang et al. 2007) and that we somehow failed to achieve this moral standard. If we are mistaken in that interpretation, then we would appreciate it if Dr. Costerton would say so. If we are not mistaken, then we would also appreciate it if Dr. Costerton would explain exactly how we failed in that regard. Our one and only motivation was very straightforward: the vast majority of Dr. Balaban?s work, at least with respect to TRAP itself, has been done with a single lineage (8325-4) of S. aureus. This lineage has significant, well-recognized problems that impact global regulatory circuits. Having demonstrated ourselves that important differences exist between strains of this lineage and clinical isolates of S. aureus (Blevins et al., 2002), we wanted to determine whether TRAP plays the same regulatory role in our clinical isolates as that reported by Balaban in 8325-4 strains. We examined this issue experimentally, including our 8325-4 as a ?control?, and published our results. It seems to us that this is in the appropriate scientific spirit of clarifying the issue, and we are unclear how Dr. Costerton or anyone else could interpret it as an attempt to ?discredit? anyone?

Dr. Costerton goes on to ask a series of questions including ?Could the disrepancies that fuel this sad debate be caused by the action of signals that curently lurk in the background as the products of some ORF of unknown function? Could it be that we are all exploring a jungle in which we don't even know what forces are operative? Could it be that we "see through a glass darkly", and that we really should help each other find the answers?? Once again, we would ask that he please explain why he views our decision to investigate this issue and publish our results as a ?sad debate? rather than attempt to answer these very questions.

In a separate letter, Dr. Medina-Acosta dismisses our peer-reviewed publication (Tsang et al. 2007) and that of Shaw et al. (2007), by stating ?the two articles in which mutagenized TRAP with no effect is claimed barely, if at all, describe how their mutagenesis was done or its effect on TRAP function in their resulting mutants?. We will let Les Shaw and George Stewart respond as they see fit, but for our part the protocols we used for mutagenesis, including the methods we used to verify our mutants, are included in our paper, and it is unclear to us what is missing from that description. Again, if Dr. Medina-Acosta believes we have failed in that regard, then we would appreciate it if he would be more specific and tell us exactly what is missing that would prevent him from repeating our experiments if he chose to do so. With respect to what Dr. Medina-Acosta considers our failure to assess ?TRAP function?, the Balaban group proposes that RIP functions by inhibiting phosphorylation of TRAP and thereby limiting activation of agr which in turn results in reduced toxin production and reduced biofilm formation. Our conclusion was that mutation of traP has no effect on expression of agr (as evidenced by the production of wild-type levels of RNAIII and hemolytic activity) or on biofilm formation in the isolates we examined (as assessed by comparison to the corresponding parent strain). In short, we assessed the most relevant phenotypes that Dr. Balaban has ascribed to TRAP, and in light of that we are unclear why Dr. Medina-Acosta would conclude that we did not examine ?TRAP function?. Moreover, Dr. Balaban asked for our mutants and we sent them to her. Because our experiments did not address the issue of whether RIP inhibits biofilm formation, we also asked Dr. Balaban if she would supply us with active RIP. She agreed, but in a subsequent exchange we were told by Dr. Balaban that ?We had sent you RIP powder a while ago by regular mail. It should have arrived ages ago?. We were also told that she did not have any more and that we should request it from ?any peptide-producing company. Just make sure they make it in an amide form on the C terminus (YSPWTNF-NH2)?. We took this advice and, having also been told that ?The source of RIP I used was from Neosystem but they are now under contractual agreements?, we had it synthesized by a company Dr. Balaban cites in one of her own papers (Genemed Synthesis Inc, as cited in Balaban et al., 2000, Peptides 21:1301-1311). Given that we are asking others to be more precise in their criticisms, we will take our own advice and note for the record that we are quoting our e-mail exchanges with Dr. Balaban directly and do not mean to imply anything other than the fact that, for whatever reason, we were not able to obtain RIP directly from Dr. Balaban.

Using the RIP we obtained from Genemed, we have begun to investigate the efficacy of RIP with respect to inhibition of biofilm formation, and our preliminary results indicate that RIP may in fact inhibit biofilm formation but only at concentrations that are far too high to be biologically relevant. In that regard, it is important to clarify what this debate is about. To our knowledge, the primary if not the only concern expressed by other investigators in the staphylococcal research community is whether RIP is relevant with respect to the S. aureus agr signaling pathway and, for our part, whether this has an impact on biofilm formation. The only conclusion we have published to date is that, if RIP does in fact function in that capacity, it must not function through TRAP since mutation of traP had no effect on expression of agr or biofilm formation in any of the strains we examined (Tsang et al., 2007). We stand by this conclusion, which says nothing about whether RIP can be synthesized and used therapeutically. These are two entirely separate issues, and they should not be confused. Perhaps Dr. Costerton has confused them as evidenced by his suggestion that we all ?stay tuned to the Springer Series on Biofilms, and watch the picture sharpen!? We were intrigued by that comment, so we went online to find this text. What we found was a text entitled ?Control of Biofilm Infections by Signal Manipulation?. (LINK While we cannot access the text itself, it is important to note that the title itself does not refer to the use of synthetic RIP in therapy but rather to the guided manipulation of an S. aureus signaling pathway, and the ability to do that using RIP is the only conclusion we are aware of that has been called into question.

For the record, Dr. Balaban is the sole editor of the Springer text and is an author on 6 of the 10 chapters listed online. In that regard it is important to address one final issue. Specifically, Dr. Balaban has indeed published a long list of papers, all of which champion some aspect of the RAP/RIP/TRAP/agr/biofilm pathway summarized above. In point of fact, a primary reason we chose to pursue these experiments is that there are also a number of publications (Beenken et al., 2003, Vuong et al., 2000, Yarwood et al., 2004, Yarwood et al., 2007) concluding that expression of agr inhibits biofilm formation. These were all published in peer-reviewed journals by independent labs both with respect to each other and Dr. Balaban?s lab, and they began appearing in the literature as early as 2000 (Vuong et al., 2000). In fact, the latest of these (Yarwood et al., 2007) concludes that agr mutants arise spontaneously in biofilms and ultimately become the dominant subpopulation. These reports are at direct odds with Dr. Balaban?s overall hypothesis. Specifically, she suggests that a primary function of RIP is inhibition of agr expression. Based on the reports cited above, this would be expected to enhance rather than inhibit biofilm formation. Dr. Costerton suggests that something may ?lurk in the background? to explain this very obvious discrepancy, and that may well be the case (again, that is why we chose to investigate the issue), but what we don?t understand is why Dr. Balaban has steadfastly refused to even acknowledge this discrepancy in any of her papers including The Scientist article in question. It will indeed be interesting to ?stay tuned? to see if she continues to ignore this issue in her Springer text. If so, then we fail to see how that will help the ?picture sharpen?.

Mark S. Smeltzer and Laura H. Tsang
Department of Microbiology and Immunology
University of Arkansas for Medical Sciences

In our experiments RIP worked as well or even better than commonly
used antibiotics. RIP and antibiotics were used the same
concentrations ranges and are synergistic to one another. These
results have been observed over the last 5 years in multiple animal
models. RIP is thus a promising molecule for the prevention and
treatments of staphylococcal infections.

Dear Bill,

I am writing this to stress that first, I completely agree ? like everyone I know who has profound knowledge on gene regulation in staphylococci ? with the scientific assessment by Dr. Novick. Second, there is no unfair treatment of Dr. Balaban as a scientist, such as implied by you and Dr. Medina-Acosta. In contrast, many staphylococcal researchers have given Dr. Balaban multiple chances to perform the experiments necessary to prove that her conclusions are valid. I have myself talked to her at conferences, suggesting the obvious controls needed to convince the field and myself. I would have wished, too, that the dispute had been settled as between you and Drs. Greenberg, Iglewski, Stoodley, and Lappin-Scott. However, unfortunately, Dr. Balaban has avoided rather than addressed the suggested experimental validation and preferred to do away with very reasonable concerns about technical flaws and missing controls in her studies only using odd discussions in her publications. As this has been going on for years, there is understandable frustration with Dr. Balaban?s behavior on the part of scientists interested in the scientific truth. In that regard, rather than attack Drs. Smeltzer, Stewart, Novick, and Shaw, I have to applaud them using their time and resources to find a final settlement to the dispute at last, although everyone knows how unrewarding the publication of negative results is. Their results unambiguously show that the RIP/RAP/TRAP concept is plainly wrong. I wish that by accepting this, like the vast majority of researchers in the field have done, resources could be saved for science that moves the field forward.

As for the frequency of mutations in the agr system, one of the reasons for the wrong conclusions on the alleged function of TRAP on agr, I have to stress that I have all important clones in my lab tested for agr functionality during every key experiment. I can only encourage all staphylococcal researchers to do so, too. This will hopefully prevent further wrong conclusions on alleged gene regulatory effects, something that has not only happened in the case of TRAP, but already several times.

All the best

Michael

I post this comment with a profound sense of sadness concerning researchers who try to drag their colleagues down, instead of opening up new insights. I am not an expert in the signaling area, but I have almost 5 decades of experience in research, and one of these experiences may be helpful in this overheated debate.

In 1998 we published a paper that described a modification of biofilm formation in Las - negative mutants of P. aeruginosa, with two giants of the signaling field (Barbara Iglewski and Pete Greenberg). This paper became an anchor of the new field of signal control of biofilm formation, so we could have become the target of small minded attacks, and colleagues could have gained bragging rights by proving us wrong. In fact two close friends, Paul Stoodley and Hilary Lappin-Scott of Exeter University, tried to repeat our experiments using different Las minus mutants and got very different results. Instead of trying for a Science paper that would refute our publication, Paul and Hilary gave us a very thoroughly annotated folder showing their contradictory data, and we puzzled over the dilemma in several wine-lubricated discussions. We could see not way out until a minor observation, that signal interactions depend on iron concentrations, emerged in the large research center that I directed. We finally understood how two teams, with similar enthusiasm and similar scientific ethics, could have gotten tangled up in a vicious fight. The moral - talk to each other decently and don't publish to discredit, because the field will clarify as more data emerges, and all will be revealed.

Bacterial signaling systems form a very complex web of interactions, and I predict that the number of systems we discover will increase exponentially with time. Could the disrepancies that fuel this sad debate be caused by the action of signals that curently lurk in the background as the products of some ORF of unknown function? Could it be that we are all exploring a jungle in which we don't even know what forces are operative? Could it be that we "see through a glass darkly", and that we really should help each other find the answers?

The gold standard will be reached if RIP helps patients, so stay tuned to the Springer Series on Biofilms, and watch the picture sharpen!

Davies, D.G., Parsek, M.R., Pearson, J.P., Iglewski, B.H., Costerton, J.W., and Greenberg, E.P. 1998. The Involvement of Cell-to-Cell Signals in the Development of a Bacterial Biofilm, Science, 280:295-298.

R.P. Novick has launched an offensive and, to say the least, unfairly discreditable qualification of N. Balaban?s research on the quorum-sensing interfering RNA-inhibiting peptide (RIP) as a questionable therapy backed by questionable science, a campaign that nothing more resumes and mirrors an over-a-decade (1990-2008) heated, yet highly deleterious, controversy affecting three sides: theirs and the reader?s.

To the reader, essentially two schools of thought on quorum-sensing in the genus Staphylococcus have emerged from the same framework. Although strangely conceived, in part, from the same seed and birthplace (Novick?s lab at NYU and the bench work of former post-doc Balaban) as twin quorum-sensing systems, today, and more than ever, one parent (Novickﾴs side) is reluctant to rest the thoughts in peace. To Novick, both schools are inconceivable. To Balaban, both are part of broader regulatory network. Notably, Novick views Balabanﾴs studies contradictory to the establishment, a field which he pioneered and thus, at all costs, so it seems, wants the reader to regard the RIP-RAP-TRAP quorum system as inexistent; an invention, he states. The evidence that the RIP-RAP-TRAP interfering operates in vitro (cell-free) and in vivo is overwhelming (see references cited by both authors).

Most of what is left in between arguments in favor and against either school is certainly not a grandiose interpretation, which both sides appear to be shy with.

The call for more independent work on RIP, as comprehensible and important as it can be for the developing of more effective therapeutics, does not necessarily equal to a lack of proof-of-concept nor contradicts Balaban?s and/or her colleagues studies, as perhaps originally intended by Novickﾴs commentary. To my understanding, as an independent source, both QS systems co-exist and at some point they even can be integrated. Balabanﾴs findings are not a contra version of the quorum-sensing establishment, but rather a parallel functional version. As I see it, the field is only benefiting for this long-lasting debate.

A serious alerting point is, however, raided by Novick: A traP mutant he tested was also an agrA mutant (Adhikari et al., 2007). The fact that agr gets randomly mutagenized poses the very interesting scientific questions of how and why is such an important global regulatory locus be readily mutagenized. Perhaps the key is with TRAP? Clearly, this should be further studied rather than used to suppress previous data. Considering the gravity of the situation and the impact even the suspicion is having on the reputation of the field as a whole, a valid question both sides should address is whether all the published studies were carried out on strains bearing such agr mutations.

I also would like to address the comment made by George Stewart regarding recent evidence to the contrary of the RIP-RAP-TRAP quorum-signaling pathway. In the heat of the present debate, Stewart?s comment, unfortunately, spills back to the cited recent publications (Shaw et al., 2007; Tsang et al., 2007). The two articles in which a mutagenized TRAP with no effect is claimed barely ,if at all, describe how their mutagenesis was done or its effect on TRAP function in their resulting mutants. If they were proven to be correct, perhaps their mutants deviate from the norm? Either way, this will be an interesting subject to study, rather than suppress.

Lastly, I personally have used synthetic RIP extensively and found it very beneficial in suppressing experimental infections. I used the peptides in concentration ranges that are typical of normally used antibiotics; no problems with that. I also used recombinant RAP to vaccinate cows, and found this approach very effective. It is hard to understand why a group is trying, so it seems, to monopolize the field, and to deter scientific and medical progress, when both are so needed.

Enrique Medina-Acosta
Laboratory of Biotechnology
Universidade Estadual do Norte Fluminense, Brazil.

References

Shaw, L.N., I.-M. Jonnson, V. K. Singh, A. Tarkowski, and G. C. Stewart. 2007. Inactivation of traP has no effect on the Agr quorum sensing system or virulence of Staphylococcus aureus. Infect. Immun. 75:4519-4527.

Tsang, L. H., S. T. Daily, E. C. Weiss, and M. S. Smeltzer. 2007. Mutation of traP in Staphylococcus aureus has no impact on expression of agr or biofilm formation. Infect Immun. 75:4528-4533.

Adhikari, RP, S. Arvidson, and R. P. Novick. 2007. A nonsense mutation in agrA accounts for the defect in agr expression and the avirulence of Staphylococcus aureus 8325-4 traP-. Infect. Immun. 75:4534-4540.

Methicillin resistant Staphylococcus aureus (MRSA) is a significant public health issue and has garnered a great deal of attention in the press. Articles such as MRSA: RIP? are likely to be read by a wide audience and as a consequence promote concepts that overwhelming evidence now indicates are incorrect. The RAP-TRAP putative quorum sensing system was reported to be an important regulator of virulence gene expression in S. aureus. However, three recent reports in the September issue of Infection and Immunity (cited below) have shown that the TRAP protein is not involved in modulating virulence gene expression or biofilm formation, and, furthermore, has no effect on the Agr quorum sensing system, a central virulence regulatory system in this organism. Furthermore, results from these studies clearly indicate that the effects reported for the TRAP-negative mutants are the result of coincidental agr mutations. Evidence for the alleged regulatory function of RAP in S. aureus has not been obtained independently of the work of Dr. Balaban and co-workers.

As for the claimed potential use of RIP as an anti-biofilm therapeutic, it needs to be stressed that many proteins (or peptides), when applied in the high concentrations of RIP in the experiments by the Balaban group, alter biofilm formation by mere physico-chemical interaction. If the biofilm effect were due to the alleged, erroneous signaling function of RIP, one would expect it to be active at lower concentrations.

Thus, the development of therapeutics based on a signaling system shown to be entirely erroneous would be, at best, a sorely misguided strategy.

Shaw, L.N., I.-M. Jonnson, V. K. Singh, A. Tarkowski, and G. C. Stewart. 2007. Inactivation of traP has no effect on the Agr quorum sensing system or virulence of Staphylococcus aureus. Infect. Immun. 75:4519-4527.

Tsang, L. H., S. T. Daily, E. C. Weiss, and M. S. Smeltzer. 2007. Mutation of traP in Staphylococcus aureus has no impact on expression of agr or biofilm formation. Infect Immun. 75:4528-4533.

Adhikari, RP, S. Arvidson, and R. P. Novick. 2007. A nonsense mutation in agrA accounts for the defect in agr expression and the avirulence of Staphylococcus aureus 8325-4 traP-. Infect. Immun. 75:4534-4540.


Michael Otto
Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases
Lindsey N. Shaw
Department of Biology, University of South Florida

Mark S. Smeltzer
Department of Microbiology and Immunology, University of Arkansas for Medical Sciences

Simon J. Foster
Department of Molecular Biology and Biotechnology, University of Sheffield

George C. Stewart
Department of Veterinary Pathobiology, University of Missouri

I would like to thank the readers for their comments. To address a few, RIP is in fact especially effective when used in combination, making commonly used antibiotics more effective. It is thus not a magic bullet but a deeply studied system with so far phenomenal results. RIP inhibits both toxin production and biofilm formation, making the bacteria less resistant to antibiotic treatment and more vulnerable to an immune response.

As for Dr. Novick?s remarks, he has made similar assertions in the past, which are not supported by the numerous peer-reviewed reports (see below) that show that RIP does indeed suppress MRSA infections and that the technology is indeed effective.

References:
Control of Biofilm Infections by Signal Manipulation. Balaban N, Ed. Springer 2008.

Costerton JW, et al. Bacterial communications in implant infections: a target for an intelligence war. Int J Artif Organs. 2007 30:757-63.

Cirioni O, et al. RNAIII-inhibiting peptide affects biofilm formation in a rat model of staphylococcal ureteral stent infection. Antimicrob Agents Chemother. 2007 51:4518-20.

Balaban N, et al. Treatment of Staphylococcus aureus biofilm infection by the quorum-sensing inhibitor RIP. Antimicrob Agents Chemother. 2007 51:2226-9.

Anguita-Alonso P, et al. RNAIII-inhibiting-peptide-loaded polymethylmethacrylate prevents in vivo Staphylococcus aureus biofilm formation. Antimicrob Agents Chemother. 2007 51:2594-6.

Ghiselli R, et al. RNAIII-inhibiting peptide in combination with the cathelicidin BMAP-28 reduces lethality in mouse models of staphylococcal sepsis. Shock. 2006 26:296-301.

Cirioni O, et al. RNAIII-inhibiting peptide significantly reduces bacterial load and enhances the effect of antibiotics in the treatment of central venous catheter-associated Staphylococcus aureus infections. J Infect Dis. 2006 193:180-6.

Yang G, et al. A novel peptide isolated from phage library to substitute a complex system for a vaccine against staphylococci infection. Vaccine. 2006 24:1117-23.

Yang G, et al. A novel peptide screened by phage display can mimic TRAP antigen epitope against Staphylococcus aureus infections. J Biol Chem. 2005 280:27431-5.

Balaban N, et al. Prevention of staphylococcal biofilm-associated infections by the quorum sensing inhibitor RIP. Clin Orthop Relat Res. 2005 437:48-54.

Giacometti A, et al. RNAIII-inhibiting peptide improves efficacy of clinically used antibiotics in a murine model of staphylococcal sepsis. Peptides. 2005 26:169-75.

Domenico P, et al. BisEDT and RIP act in synergy to prevent graft infections by resistant staphylococci. Peptides. 2004 25:2047-53.

Dell'Acqua G, et al. Suppression of drug-resistant Staphylococcal Infections by the quorum-sensing inhibitor RNAIII-inhibiting peptide. J Infect Dis. 2004 190:318-20.

Balaban N, et al. A chimeric peptide composed of a dermaseptin derivative and an RNA III-inhibiting peptide prevents graft-associated infections by antibiotic-resistant staphylococci. Antimicrob Agents Chemother. 2004 48:2544-50.

Ghiselli R, et al. RNAIII-inhibiting peptide and/or nisin inhibit experimental vascular graft infection with methicillin-susceptible and methicillin-resistant Staphylococcus epidermidis. Eur J Vasc Endovasc Surg. 2004 27:603-7.

Cirioni O, et al. Prophylactic efficacy of topical temporin A and RNAIII-inhibiting peptide in a subcutaneous rat Pouch model of graft infection attributable to staphylococci with intermediate resistance to glycopeptides. Circulation. 2003 108:767-71.

Giacometti A, et al. RNA III inhibiting peptide inhibits in vivo biofilm formation by drug-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2003 47:1979-83.

Balaban N, et al. Use of the quorum-sensing inhibitor RNAIII-inhibiting peptide to prevent biofilm formation in vivo by drug-resistant Staphylococcus epidermidis.J Infect Dis. 2003 187:625-30.

Ribeiro PD, et al. Treatment efficacy of the lead RNAIII-inhibiting peptide YSPWTNF-NH2 in acquired Staphylococcus aureus sepsis: a histopathological assessment. Peptides. 2003 24:1829-36.

Yang G, et al. Inhibition of Staphylococcus aureus pathogenesis in vitro and in vivo by RAP-binding peptides. Peptides. 2003 24:1823-8.

Balaban N, et al. Prevention of diseases caused by Staphylococcus aureus using the peptide RIP. Peptides. 2000 21:1301-11.

Balaban N, et al. Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus. Science. 1998 280:438-40.

In your preface to the MRSA: RIP? article, you note that you're ''publishing the piece early online in an extended version to spark discussion of the science.'' Indeed, you have --- In fact, there has been much ''discussion'' and controversy about this ''science'' over the last several years. However, Andrea Gawrylewski's article has noted none of this. The article celebrates a very extensive but deeply flawed body of work authored entirely by Balaban and her colleagues. Despite its claimed importance, no independent confirmation of any of it has yet been published.
This work was initiated in my lab when I asked Naomi Balaban, then a new post-doc, to analyze staphylococcal culture supernatants for substances which would affect the expression of agr, an important regulator of bacterial virulence. Balaban confirmed the existence of an activator, which she called RAP, and identified a peptide inhibitor, which she called RIP, in the supernatants. However, she misidentified the activator as a protein (it is actually a small peptide ? see below), generated an imaginary sequence for the inhibitory peptide (8), and has tenaciously clung to both misconceptions ever since.
agr is conserved throughout the staphylococci and also occurs in various other bacteria. There is a near-universally accepted model, holding that the agr locus consists of a two-component signal transduction module plus two genes that combine to produce a thiolactone-containing autoinducing peptide (AIP) that activates its own receptor (AgrC) and inhibits the agr receptors of other strains (6). agr acts by inducing a regulatory RNA, RNAIII (7), that controls expression of most of the virulence genes (7). The Balaban group has assiduously propounded a nouveau model in which the protein RAP (ribosomal protein L2) activates a second protein, TraP, which activates RNAIII expression, independently of the agr signaling module. RIP blocks the RAP-TraP pathway, interfering with rnaIII expression and thereby inhibiting biofilm formation and attenuating staphylococcal infections.
Unfortunately, all three components of this RIP-RAP-TraP model are fatally flawed:
RIP. Several labs have synthesized RIP and found it to have no significant effect on agr function at concentrations up to 50 micromolar (8). In fact, data published by Balaban and coworkers are consistent with this, failing to show significant inhibition of RNAIII expression by RIP. Fig. 2B from Balaban, et al. (2) shows that RIP, even at a concentration of 150 micromolar (point on the graph corresponding to 5 micrograms), causes no more than 30% inhibition. At this concentration, the very modest inhibition by RIP is almost certainly non-specific, could easily represent an overall effect on bacterial metabolism, and would be most unlikely to affect the expression of RNAIII-regulated genes directly.
So where did this ''RIP'' come from, and what is the basis for the curious claim that it inhibits agr signaling?
When Naomi Balaban joined my lab as a post-doc, she was assigned to confirm, as noted above, our previous finding of a soluble activator of agr signaling. All but one of 15 strains that were tested produced such an activator, whereas the exceptional strain, RN833, produced a peptide inhibitor of agr activation in our standard lab strain. Although this peptide turned out to be the agr-encoded autoinducing peptide (AIP) of strain RN833, we initially (and naively) designated it ''RIP'' (for RNAIII-inhibiting peptide). Balaban later obtained the sequence, YSP-TNF, for the peptide and, given that the peptide sequencing methods then being used did not differentiate cysteine or tryptophan, arbitrarily inserted tryptophan (W) for the indeterminate central amino acid.
This was difficult to understand since gene sequencing and mass spectroscopy had already shown that staphylococcal AIPs contain a cysteine in that position (4) (5). Subsequent sequencing of the RN833 AIP gene (agrD) revealed that the central amino acid is in fact, cysteine and that the AIP sequence is YSP**C**TNF**F**, a typical staphylococcal AIP (3), and this was confirmed by biochemical testing (8). Accordingly, Balaban and coworkers insist that YSP**W**TNF is not the AIP after all, but is encoded (8) and produced by RN833, along with the AIP, claiming that the two are unrelated despite their highly similar sequences. To support their claims, Balaban, et al. have developed the RIP-RAP-TraP model. Yet they have never confirmed (e.g., by mass spectroscopy) that RN833 produces YSP**W**TNF ? although at the concentrations that they use for testing, it should be readily detectable - whereas we have shown that RN833 produces only a single agr-activating/inhibiting substance, the classical thiolactone-containing YSP**C**TNF**F** (8), which is readily detectable at the very low concentration needed for full function (~50 nanomolar).
Neither our group nor any other independent group has analyzed the effects of (RIP) YSP**W**TNF on biofilms. However, there is a large body of literature suggesting that agr-negatives form more robust biofilms than agr-positives (11) (12) (13). If RIP were acting to block agr functioning, then it would enhance rather than inhibit biofilm formation. Additionally, RIP has recently been used for biofilm inhibition at concentrations much lower than those shown by several labs to have no effect on agr signaling, as noted above. Therefore, it is clear that if RIP were to have any effect on biofilms it would have to be by some other mechanism.
RAP. (RNAIII-activating protein). Balaban et al. have reported that ribosomal protein L2 , an essential intracellular protein, is also secreted and in its secreted form is an agr-RNAIII activator (RAP). As is the case with RIP, there has been no independent confirmation of the reported signaling role of this protein. In fact, contrary to the report that RAP is secreted by an agr-negative strain (2), we have failed to demonstrate any agr-RNAIII activator in culture supernatants of this same strain (8).
TraP. Balaban et al. have never provided genetic confirmation of the supposed linkage between traP and its purported phenotype. Three recently reported studies, independently and carefully performed, have demonstrated conclusively that traP mutations have no phenotype in otherwise wild-type strains. In other words, TraP does not regulate staphylococcal virulence or agr signaling (1) (9) (10). One of the studies showed that the virulence defect of the Balaban traP mutant is not complemented by the cloned traP but, instead, is complemented by the cloned agrA, the agr response regulator gene (1). This result is fully accounted for by the presence of an unrecognized inactivating mutation in the agrA gene in the Balaban traP mutant strains, as revealed by sequencing (1).
It is clear from the Gawrylewski article that by continuing to promulgate this model, Balaban and coworkers completely disregard published literature that does not support their claims.
REFERENCES
1. Adhikari, R. P., S. Arvidson, and R. P. Novick. 2007. A nonsense mutation in agrA accounts for the defect in agr expression and the avirulence of Staphylococcus aureus 8325-4 traP::kan. Infect Immun 75:4534-40.
2. Balaban, N., T. Goldkorn, R. T. Nhan, L. B. Dang, S. Scott, R. M. Ridgley, A. Rasooly, S. C. Wright, J. W. Larrick, R. Rasooly, and J. R. Carlson. 1998. Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus. Science 280:438-40.
3. Dufour, P., S. Jarraud, F. Vandenesch, T. Greenland, R. P. Novick, M. Bes, J. Etienne, and G. Lina. 2002. High genetic variability of the agr locus in Staphylococcus species. J Bacteriol 184:1180-6.
4. Ji, G., R. Beavis, and R. P. Novick. 1997. Bacterial interference caused by autoinducing peptide variants. Science 276:2027-2030.
5. Ji, G., R. C. Beavis, and R. P. Novick. 1995. Cell density control of staphylococcal virulence mediated by an octapeptide pheromone. Proc. Natl. Acad. Sci. USA 92:12055-12059.
6. Novick, R. P. 2003. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol. Microbiol. 48:1429-49.
7. Novick, R. P., R.P., H. F. Ross, S. J. Projan, J. Kornblum, B. Kreiswirth, and S. Moghazeh. 1993. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J. 12:3967-3975.
8. Novick, R. P., Ross, H.F., Figueiredo, A.M.S., Abramochkin, G., Muir, T. 2000. Activation and inhibition of the Staphylococcal AGR system. Science 287:391a.
9. Shaw, L. N., I. M. Jonnson, V. K. Singh, A. Tarkowski, and G. C. Stewart. 2007. Inactivation of traP has no effect on the Agr quorum sensing system or virulence of Staphylococcus aureus. Infect Immun.
10. Tsang, L. H., S. T. Daily, E. C. Weiss, and M. S. Smeltzer. 2007. Mutation of traP in Staphylococcus aureus has no impact on expression of agr or biofilm formation. Infect Immun.
11. Vuong, C., C. Gerke, G. A. Somerville, E. R. Fischer, and M. Otto. 2003. Quorum-sensing control of biofilm factors in Staphylococcus epidermidis. J Infect Dis 188:706-18.
12. Vuong, C., H. L. Saenz, F. Gotz, and M. Otto. 2000. Impact of the agr quorum-sensing system on adherence to polystyrene in Staphylococcus aureus. J Infect Dis 182:1688-93.
13. Yarwood, J. M., D. J. Bartels, E. M. Volper, and E. P. Greenberg. 2004. Quorum sensing in Staphylococcus aureus biofilms. J Bacteriol 186:1838-50.

When will we learn that there is seldom any magic bullet? Sure,this approach may work. It may work for years. But eventually, staph will adapt. It's almost inevitable.

Unless we alter the way we treat such problems, this "cure" will go the same way antibiotics are headed now.

While the article indicates that a molecular biological approach may ultimately be successful in controlling MERSA, conceptually another strategy may yield faster results. The toxic peptides produced by the S. aureus in question are N-terminal fMet moieties. Some time ago smaller peptides such as fMet-Leu-Phe were found to be potent leukoattractants. Although these were not tested for their lytic effects upon phagocytes, we did not observe any. However, antagonistic peptides were developed, among which was tBoc-L-Phe-D-Leu-L-Phe-D-Leu-L-Phe. The IC-50 for this is ~ 5x10-6M. Presumably this compound could compete with the MERSA peptides for the cognate receptor on neutrophils, thus preventing the bacterial products from lysing those cells. Some comments: the antagonist contains 'unnatural' amino acids which may make it less susceptible to proteolytic degradation; these compounds have not been tested for problematic effects in vivo; and the potency of this specific agent leaves something to be desired. However, if such hurdles are overcome, the approach which focuses upon the phagocyte seems worth exploring. The leukocyte would not be 'disabled' from responding to bacterial infection since it has other receptors, such as for C5a, to be activated in its host defense functions.

Virulent staph enter an open skin puncture in rather limited numbers and, unless promptly inactivated or killed locally, will colonize the wound and establish highly resistant encapsulated cell clusters known as biofilms.
Once formed, such biofilms tend to be resistant to most small molecule antimicrobials, probably also including the proposed large molecule RIP and antibody therapies. The proposed peptide based therapies furthermore may have short half lives due to proteolytic in vivo destruction, hence their effectiveness may well be limited, except as a possible last resort therapy in disseminated toxemia.
I believe the key to reducing MERSA fatalities is immediate self treatment of any small wound with any of the commonly available antimicrobials, such as iodine (povidone), hydrogen peroxide, sucrose (or honey)-povidone, followed by applying an adhesive strip. If inflammation or spread of infection are observed after about one day, a visit to the doctor is advisable.
Longer term, prophylaxis via an anti-staph vaccine appears preferable, but is probably further down the road.
HR, M.S. chemistry, consultant