Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors

Andresen, Liis; Varik, Vallo; Tozawa, Yuzuru; Jimmy, Steffi; Lindberg, Stina; Tenson, Tanel; Hauryliuk, Vasili

doi:10.1038/srep35824

Cited by 19 publications

(25 citation statements)

References 46 publications

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“…Further, bacteria exposed to hostile conditions such as nutritional starvation and other kinds of stresses induce the stringent response, which is mediated by (p)ppGpp and helps the bacteria survive under such conditions (10). Antimicrobials that target stress induced stringent response pathways are very few; thus, such an approach offers a unique possibility (22). …”

Section: Discussionmentioning

confidence: 99%

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria

Syal

Flentie

Bhardwaj

et al. 2017

Antimicrob Agents Chemother

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Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by Rel Msm in the presence of designed inhibitors in a dosedependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis. The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.

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Section: Discussionmentioning

confidence: 99%

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria

Syal

Flentie

Bhardwaj

et al. 2017

Antimicrob Agents Chemother

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“…As we devote our future efforts to unraveling the molecular mechanisms linking (p)ppGpp to metal homeostasis, the findings presented in this study will have immediate and broad implications. The prominent role of (p)ppGpp regulation in the virulence of a number of bacterial pathogens and in antibiotic persistence has been extensively documented (3)(4)(5), and compounds that interfere with (p)ppGpp signaling have been identified (45,46). It will be interesting to learn whether the associations among (p)ppGpp, metal homeostasis, and virulence in E. faecalis also occur in other bacterial pathogens and whether the associations can be further explored to develop new anti-infective approaches.…”

Section: Linkage Of (P)ppgpp and Metal Homeostasismentioning

confidence: 99%

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

2017

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In Enterococcus faecalis, the regulatory nucleotides pppGpp and ppGpp, collectively, (p)ppGpp, are required for growth in blood, survival within macrophages, and virulence. However, a clear understanding of how (p)ppGpp promotes virulence in E. faecalis and other bacterial pathogens is still lacking. In the host, the essential transition metals iron (Fe) and manganese (Mn) are not readily available to invading pathogens because of a host-driven process called nutritional immunity. Considering its central role in adaptation to nutritional stresses, we hypothesized that (p)ppGpp mediates E. faecalis virulence through regulation of metal homeostasis. Indeed, supplementation of serum with either Fe or Mn restored growth and survival of the Δrel ΔrelQ [(p)ppGpp 0 ] strain to wild-type levels. Using a chemically defined medium, we found that (p)ppGpp accumulates in response to either Fe depletion or Mn depletion and that the (p)ppGpp 0 strain has a strong growth requirement for Mn that is alleviated by Fe supplementation. Although inactivation of the nutrient-sensing regulator codY restored some phenotypes of the (p)ppGpp 0 strain, transcriptional analysis showed that the (p)ppGpp/CodY network does not promote transcription of known metal transporters. Interestingly, physiologic and enzymatic investigations suggest that the (p)ppGpp 0 strain requires higher levels of Mn in order to cope with high levels of endogenously produced reactive oxygen species (ROS). Because (p)ppGpp mediates antibiotic persistence and virulence in several bacteria, our findings have broad implications and provide new leads for the development of novel therapeutic and preventive strategies against E. faecalis and beyond. KEYWORDS (p)ppGpp, Enterococcus, manganese, metal homeostasis, nutritional immunity, oxidative stress E nterococcus faecalis is a common member of the human gut microbiota but also a leading cause of a number of hospital-acquired infections such as endocarditis and surgical wound and urinary tract infections (1). The association of this opportunistic pathogen with disease relies on its exceptional resilience, which allows it to prevail in the unfavorable hospital setting while providing a competitive advantage over other bacteria during both infection and treatment.Among the most prominent regulators involved in bacterial adaptation to stress are the nucleotide second messengers guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), collectively, (p)ppGpp, best known as the effectors of the stringent response (SR) (2). While initially discovered to accumulate in response to amino acid starvation, (p)ppGpp has also been shown to accumulate in response to restrictions in carbon, fatty acids, and iron, as well as in response to other nonnutritional stresses (3). During the SR, (p)ppGpp accumulation triggers transcriptional alterations that lead to general repression of rapid growth and simultaneous activation of stress survival, nutrient uptake, and biosynthesis pathways. In addition to its involvement in...

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“…Um peptídeo anti-biofilme (peptídeo 1018) foi proposto como tendo como alvo (p)ppGpp, levando à sua degradação in vivo, embora possuindo também um forte efeito bactericida (de la Fuente-Núñez et al, 2014). Todavia, nem a relacina nem o peptídeo 1018 passaram no critério de seleção de uma triagem para antagonistas de (p)ppGpp baseada no fenótipo de auxotrofia, que se baseava na expectativa de que uma molécula capaz de impedir a síntese de (p)ppGpp deveria tornar uma linhagem selvagem auxotrófica (Andresen et al, 2016b). Adicionalmente, o peptídeo 1018 foi testado em mais ensaios, e sua especificidade por (p)ppGpp, assim como sua capacidade de diminuir seus níveis in vivo mostraram-se questionáveis (Andresen et al, 2016a (Syal et al, 2017).…”

Section: Resposta Estringente Como Alvo De Novas Drogasunclassified

“…Nesses casos, mesmo pequenos incrementos de (p)ppGpp ou então a manutenção de níveis basais de (p)ppGpp são capazes de evitar a perda de viabilidade, a auxotrofia e impedir o acúmulo de H2O2 (Gaca et al, 2013;Traxler et al, 2008;Gaca et al, 2015) por exemplo. Isso pode explicar porque a droga relacina, apesar de ser capaz de reduzir parcialmente os níveis in vivo de (p)ppGpp, é incapaz de gerar um fenótipo de auxotrofia em células de B. subtilis (Andresen et al, 2016b). Consequentemente, ainda se carece da descoberta de inibidores mais potentes da resposta estringente.…”

Section: Resposta Estringente Como Alvo De Novas Drogasunclassified

“…aureus e MRSA (Gardete & Tomasz, 2014;Dordel et al, 2014 Por fim, buscou-se também avaliar se a salicilanilida possuía a capacidade de gerar o fenótipo de auxotrofia na cepa ∆RelQ ∆RelP, uma vez que é sabido que cepas incapazes de produzir (p)ppGpp não crescem em meios mínimos não suplementados com todos os aminoácidos Andresen et al, 2016b). Observou-se, todavia, que a salicilanilida não foi capaz de gerar o fenótipo de auxotrofia na cepa ∆RelQ ∆RelP; de modo contrário, aparentou aumentar o crescimento, ao passo que a cepa ppGpp(0) foi incapaz de crescer no meio (Figura 31).…”

Section: Avaliação Da Droga Salicilanilida Como Potencial Inibidor Daunclassified

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Estudos da resposta estringente de <i>Bacillus subtilis</i> e busca por pequenas moléculas moduladoras de RelA

Pulschen¹

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Auxotrophy-based High Throughput Screening assay for the identification of Bacillus subtilis stringent response inhibitors

Cited by 19 publications

References 46 publications

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria

Association of Metal Homeostasis and (p)ppGpp Regulation in the Pathophysiology of Enterococcus faecalis

Estudos da resposta estringente de <i>Bacillus subtilis</i> e busca por pequenas moléculas moduladoras de RelA

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