scholarly journals Identification of Effectors of Synergistic Lethality in Candida albicans-Staphylococcus aureus Polymicrobial Intra-abdominal Infection

2021 ◽  
Author(s):  
◽  
Olivia Todd ◽  
Keyword(s):  
Staphylococcus Aureus ◽  
Transcription Factor ◽  
Candida Albicans ◽  
Candida Species ◽  
Toxin Production ◽  
Extracellular Ph ◽  
Abdominal Infection ◽  
Intra Abdominal Infection

Candida albicans, an opportunistic fungal pathogen, and Staphylococcus aureus, a ubiquitous pathogenic bacterium, are among the most prevalent causes of nosocomial infections and cause severe morbidity and mortality. Moreover, they are frequently coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Relatively little is known about the complex interactions and signaling events that occur between microbes and even less so how microbial “cross-talk” shapes human health and disease. Using a murine model of polymicrobial intra-abdominal infection (IAI), we have previously shown that coinfection with C. albicans and S. aureus leads to synergistic lethality whereas monomicrobial infection is nonlethal. Therefore, we aimed to identify staphylococcal virulence determinants that drive lethal synergism in polymicrobial IAI. Using the toxigenic S. aureus strain JE2, we observed that co-infection with C. albicans led to a striking 80-100% mortality rate within 20 h p.i while monomicrobial infections were non-lethal. Use of a GFP-P3 promoter S. aureus reporter strain revealed enhanced activation of the staphylococcal agr quorum sensing system during in vitro polymicrobial versus monomicrobial growth. Analyses by qPCR, Western blot, and toxin functional assays confirmed enhanced agr-associated gene transcription and increases in secreted α- and δ-toxins. C. albicans-mediated elevated toxin production and hemolytic activity was determined to be agrA-dependent and genetic knockout and complementation of hla identified ⍺-toxin as the key staphylococcal virulence factor driving lethal synergism. Analysis of mono- and polymicrobial infection 8 h p.i. demonstrated equivalent bacterial burden in the peritoneal cavity, but significantly elevated levels of α-toxin (3-fold) and the eicosanoid PGE2 (4-fold) during co-infection. Importantly, prophylactic passive vaccination using the monoclonal anti-⍺-toxin antibody MEDI4893* led to significantly improved survival rates as compared to treatment with isotype control antibody. Collectively, these results define α-toxin as an essential virulence determinant during C. albicans-S. aureus IAI and describe a novel mechanism by which a human pathogenic fungus can augment the virulence of a highly pathogenic bacterium in vivo. We next sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal ⍺-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced ⍺-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and ⍺-toxin production. By using a C. albicans mutant deficient in alkalinization (stp2Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species (C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. krusei) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and ⍺-toxin production during coculture. Overall, we demonstrated that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system. Finally, we correlated ⍺-toxin production with significant increases in biomarkers of liver and kidney damage during coinfection and determined that functional toxin was required for morbidity and mortality. We next sought to determine the candidal effector(s) mediating this enhanced virulence by employing an unbiased screening approach. C. albicans transcription factor mutants were evaluated for their ability to induce S. aureus agr activation in polymicrobial culture. Incredibly, we identified several mutants that displayed defects in augmenting S. aureus agr activity in vitro. Two of the mutants failed to completely synergize with S. aureus in vivo and further analysis revealed the necessity of the uncharacterized C. albicans transcription factor, ZCF13, in driving enhanced toxin production both in vitro and in vivo. Collectively, we identified a novel effector by which C. albicans augments S. aureus virulence and identified a potential mechanism of fungal-bacterial lethal synergism.

scholarly journals Candida albicans Impacts Staphylococcus aureus Alpha-Toxin Production via Extracellular Alkalinization

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Olivia A. Todd ◽  
Mairi C. Noverr ◽  
Brian M. Peters
Keyword(s):  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Quorum Sensing ◽  
Bacterial Pathogen ◽  
Toxin Production ◽  
Extracellular Ph ◽  
Morbidity And Mortality ◽  
Alpha Toxin ◽  
Content Type

ABSTRACT Candida albicans and Staphylococcus aureus are common causes of nosocomial infections with severe morbidity and mortality. Murine polymicrobial intra-abdominal infection (IAI) with C. albicans and S. aureus results in acute mortality dependent on the secreted cytolytic effector alpha-toxin. Here, we confirmed that alpha-toxin is elevated during polymicrobial growth compared to monomicrobial growth in vitro. Therefore, this study sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal alpha-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced alpha-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and alpha-toxin production. Modulation of the pH could predictably attenuate or activate agr activity during coculture. By using a C. albicans mutant deficient in alkalinization (stp2Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species (C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. krusei) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and alpha-toxin production during coculture. Overall, we demonstrate that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system. IMPORTANCE Candida albicans and Staphylococcus aureus are commonly coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Thus, they represent a significant cause of nosocomial morbidity and mortality. Yet how these organisms behave in the context of polymicrobial growth remains poorly understood. In this work, we set out to determine the mechanism by which activation of the staphylococcal agr quorum sensing system and production of its major virulence effector alpha-toxin is enhanced during coculture with C. albicans. Surprisingly, we likely ruled out that a secreted candidal factor drives this process. Instead, we demonstrated that alkalinization of the extracellular milieu by C. albicans and other Candida species correlated with elevated agr activity. Thus, we propose a mechanism where modulation of the extracellular pH by fungal opportunists can indirectly alter virulence of a bacterial pathogen. Uncovering molecular events that drive interkingdom pathogenicity mechanisms may enhance surveillance and treatment for devastating polymicrobial infections.

scholarly journals Candida/Staphylococcal Polymicrobial Intra-Abdominal Infection: Pathogenesis and Perspectives for a Novel Form of Trained Innate Immunity

2019 ◽  
Vol 5 (2) ◽  
pp. 37 ◽  
Author(s):  
Shannon K. Esher ◽  
Paul L. Fidel ◽  
Mairi C. Noverr
Keyword(s):  
Innate Immunity ◽  
Candida Albicans ◽  
Bacterial Species ◽  
Suppressor Cells ◽  
In Vivo Models ◽  
Abdominal Infection ◽  
Intra Abdominal Infection ◽  
And Function

Polymicrobial sepsis is difficult to diagnose and treat and causes significant morbidity and mortality, especially when fungi are involved. In vitro, synergism between Candida albicans and various bacterial species has been described for many years. Our laboratory has developed a murine model of polymicrobial intra-abdominal infection with Candida albicans and Staphylococcus aureus, demonstrating that polymicrobial infections cause high levels of mortality, while monoinfections do not. By contrast, closely related Candida dubliniensis does not cause synergistic lethality and rather provides protection against lethal polymicrobial infection. This protection is thought to be driven by a novel form of trained innate immunity mediated by myeloid-derived suppressor cells (MDSCs), which we are proposing to call “trained tolerogenic immunity”. MDSC accumulation has been described in patients with sepsis, as well as in in vivo sepsis models. However, clinically, MDSCs are considered detrimental in sepsis, while their role in in vivo models differs depending on the sepsis model and timing. In this review, we will discuss the role of MDSCs in sepsis and infection and summarize our perspectives on their development and function in the spectrum of trained innate immune protection against fungal-bacterial sepsis.

scholarly journals Evaluation du potentiel antimicrobien et de la toxicité des extraits de Jatropha multifida Linn, (Euphorbiaceae)

2020 ◽  
Vol 151 ◽  
pp. 15550-15558
Author(s):  
Amégninou Agban ◽  
Yao Hoekou ◽  
Passimna Pissang ◽  
Tchadjobo Tchacondo ◽  
Komlan Batawila
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Candida Albicans ◽  
Aqueous Extract ◽  
E Coli ◽  
Toxicological Studies ◽  
Jatropha Multifida

Objectif : L’objectif de ce travail était d’évaluer in vitro l’activité antimicrobienne des extraits de feuilles et tige de Jatropha multifida sur la croissance de Candida albicans, Escherichia coli et Staphylococcus aureus, puis d’évaluer in vivo la toxicité de cette plante. Méthodologie et résultats : Les méthodes de diffusion en milieu gélosé et de microdilution en milieu liquide ont été utilisées pour évaluer l’effet antimicrobien. Une étude en subaigüe était réalisée afin d’explorer les effets toxiques de l’extrait aqueux des feuilles. Les résultats des tests antimicrobiens montrent une activité des extraits de feuilles et tige de J. multifida sur la croissance des souches utilisées avec des diamètres de zones d’inhibition allant de 8 à 25 mm et des concentrations minimales inhibitrices (CMI) variant de 0,039 mg/mL à 1,25 mg/mL à l’exception des souches de E. coli qui sont résistantes aux extraits de la tige. L’administration en subaigüe de l’extrait aqueux des feuilles de J. multifida à la dose de 600 mg/kg entraîne une perte significative de poids chez les souris. Conclusion et applications des résultats : Les extraits aqueux, éthanolique et hydroéthanolique des feuilles et tige de J. multifida possèdent d’activité antimicrobienne et pourraient être utilisés dans le traitement des Candidoses à C. albicans et des infections à S. aureus. Mais l’essai de toxicité subaigüe montre que l’extrait aqueux de la plante serait toxique. Des études toxicologiques approfondies restent donc nécessaires sur ces extraits afin de mieux élucider leur inocuité. Mots-clés : Jatropha multifida, extraits de feuilles et de tige, activités antifongique et antibactérienne, toxicité. Agban et al., J. Appl. Biosci. 2020 Evaluation du potentiel antimicrobien et de la toxicité des extraits de Jatropha multifida Linn, (Euphorbiaceae) 15551 Evaluation of antimicrobial potential and toxicity of Jatropha multifida Linn, (Euphorbiaceae) extracts ABSTRACT Objective: The objective of this study was to evaluate in vitro the antimicrobial activity of leaves and stem of Jatropha multifida extracts against Candida albicans, Escherichia coli and Staphylococcus aureus, and then to evaluate in vivo the toxicity of this plant. Methodology and Results: The agar well-diffusion and the NCCLS broth microdilution methods were used to assess the antimicrobial effect. A subacute study was carried out to explore the toxic effects of the aqueous extract of the leaves. The results of the antimicrobial tests show an activity of the extracts of leaves and stems of J. multifida on the growth of the strains used with diameters of inhibitory zones ranging from 8 to 25 mm and minimum inhibitory concentrations (MIC) varying from 0.039 mg/mL to 1.25 mg/mL exception E. coli strains which are resistant to extracts from the stem. Subacute administration of the aqueous extract of the leaves of J. multifida at a dose of 600 mg/kg leads to a significant loss of weight in the mice. Conclusion and application of findings : The aqueous, ethanolic and hydroethanolic extracts of the leaves and stem of J. multifida have antimicrobial activity and could be used in the treatment of Candidiasis and bacterial infections due respectively to C. albicans and S. aureus. But the subacute toxicity test shows that the aqueous extract of the plant would be toxic. Extensive toxicological studies therefore remain necessary on these extracts in order to better elucidate their safety. Keywords: Jatropha multifida extracts of leaves and stem, antifungal and antibacterial activities, toxicity

scholarly journals The 5′ NAD Cap of RNAIII Modulates Toxin Production in Staphylococcus aureus Isolates

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
Hector Gabriel Morales-Filloy ◽  
Yaqing Zhang ◽  
Gabriele Nübel ◽  
Shilpa Elizabeth George ◽  
Natalya Korn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Secondary Structure ◽  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Toxin Production ◽  
Dual Function ◽  
Content Type ◽  
The Stability

ABSTRACT Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5′ ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here, we report the occurrence of several NAD-RNAs in the opportunistic pathogen Staphylococcus aureus. Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium’s physiology, was found to be 5′ NAD capped in a range from 10 to 35%. NAD incorporation efficiency into RNAIII was found to depend in vivo on the −1 position of the P3 promoter. An increase in RNAIII’s NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects seem to be caused neither by changes in RNAIII’s secondary structure nor by a different translatability upon NAD attachment, as indicated by unaltered patterns in in vitro chemical probing and toeprinting experiments. Even though we did not observe any effect of this modification on RNAIII’s secondary structure or translatability in vitro, additional unidentified factors might account for the modulation of exotoxins in vivo. Ultimately, the study constitutes a step forward in the discovery of new roles of the NAD molecule in bacteria. IMPORTANCE Numerous organisms, including bacteria, are endowed with a 5′ NAD cap in specific RNAs. While the presence of the 5′ NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5′ NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating the bacterium’s virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

scholarly journals Oral Administration of the Broad-Spectrum Antibiofilm Compound Toremifene Inhibits Candida albicans and Staphylococcus aureus Biofilm FormationIn Vivo

2014 ◽  
Vol 58 (12) ◽  
pp. 7606-7610 ◽  
Author(s):  
Kaat De Cremer ◽  
Nicolas Delattin ◽  
Katrijn De Brucker ◽  
Annelies Peeters ◽  
Soña Kucharíková ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Broad Spectrum ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Candida Krusei ◽  
Content Type ◽  
And Staphylococcus Aureus

ABSTRACTWe here report on thein vitroactivity of toremifene to inhibit biofilm formation of different fungal and bacterial pathogens, includingCandida albicans,Candida glabrata,Candida dubliniensis,Candida krusei,Pseudomonas aeruginosa,Staphylococcus aureus, andStaphylococcus epidermidis. We validated thein vivoefficacy of orally administered toremifene againstC. albicans and S. aureusbiofilm formation in a rat subcutaneous catheter model. Combined, our results demonstrate the potential of toremifene as a broad-spectrum oral antibiofilm compound.

scholarly journals Mechanism of Action of a Prostaglandin E2 Receptor Antagonist with antimicrobial activity against Staphylococcus aureus

2020 ◽  
Author(s):  
Elizabeth A. Lilly ◽  
Mélanie A. C. Ikeh ◽  
Paul L. Fidel ◽  
Mairi C. Noverr
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Receptor Antagonist ◽  
Mechanism Of Action ◽  
Atp Production ◽  
Direct Growth ◽  
Prostaglandin E2 Receptor ◽  
Intra Abdominal Infection

AbstractOur laboratory recently reported that the EP4 receptor antagonist, L-161,982, had direct growth-inhibitory effects on Staphylococcus aureus in vitro and in vivo, reducing microbial burden and providing significant protection against lethality in models of S. aureus monomicrobial and polymicrobial intra-abdominal infection. This antimicrobial activity was observed with both methicillin-sensitive and methicillin-resistant S. aureus (MRSA), as well as other Gram-positive bacteria. The antimicrobial activity of L-161,982 was independent of EP4 receptor inhibitory activity. In this study, we investigated the mechanism of action (MOA) of L-161,982, which contains a sulfonamide functional group. However, results demonstrate L-161,982 does not affect folate synthesis (sulfonamide MOA), oxidative stress, or membrane permeability. Instead, our results suggest that the inhibitor works via effects on inhibition of the electron transport chain (ETC). Similar to other ETC inhibitors, L-161,982 exposure results in a small colony size variant phenotype and inhibition of pigmentation, as well as significantly reduced hemolytic activity, and ATP production. In addition, L-161,982 potentiated the antimicrobial activity of another ETC inhibitor and inhibition was partially rescued by supplementation with nutrients required for ETC auxotrophs. Taken together, these findings demonstrate that L-161,982 exerts antimicrobial activity against MRSA via inhibition the ETC, representing a new member of a potentially novel antimicrobial drug class.

scholarly journals Concurrent Local Delivery of Diflunisal Limits Bone Destruction but Fails To Improve Systemic Vancomycin Efficacy during Staphylococcus aureus Osteomyelitis

2020 ◽  
Vol 64 (7) ◽  
Author(s):  
Thomas J. Spoonmore ◽  
Caleb A. Ford ◽  
Jacob M. Curry ◽  
Scott A. Guelcher ◽  
James E. Cassat
Keyword(s):  
Staphylococcus Aureus ◽  
Adjunctive Therapy ◽  
Bone Destruction ◽  
Standard Of Care ◽  
Quorum Quenching ◽  
Toxin Production ◽  
Local Drug Delivery ◽  
Content Type

ABSTRACT Staphylococcus aureus osteomyelitis is a debilitating infection of bone. Treatment of osteomyelitis is impaired by the propensity of invading bacteria to induce pathological bone remodeling that may limit antibiotic penetration to the infectious focus. The nonsteroidal anti-inflammatory drug diflunisal was previously identified as an osteoprotective adjunctive therapy for osteomyelitis, based on the ability of this compound to inhibit S. aureus quorum sensing and subsequent quorum-dependent toxin production. When delivered locally during experimental osteomyelitis, diflunisal significantly limits bone destruction without affecting bacterial burdens. However, because diflunisal’s “quorum-quenching” activity could theoretically increase antibiotic recalcitrance, it is critically important to evaluate this adjunctive therapy in the context of standard-of-care antibiotics. The objective of this study is to evaluate the efficacy of vancomycin to treat osteomyelitis during local diflunisal treatment. We first determined that systemic vancomycin effectively reduces bacterial burdens in a murine model of osteomyelitis and identified a dosing regimen that decreases bacterial burdens without eradicating infection. Using this dosing scheme, we found that vancomycin activity is unaffected by the presence of diflunisal in vitro and in vivo. Similarly, locally delivered diflunisal still potently inhibits osteoblast cytotoxicity in vitro and bone destruction in vivo in the presence of subtherapeutic vancomycin. However, we also found that the resorbable polyester urethane (PUR) foams used to deliver diflunisal serve as a nidus for infection. Taken together, these data demonstrate that diflunisal does not significantly impact standard-of-care antibiotic therapy for S. aureus osteomyelitis, but they also highlight potential pitfalls encountered with local drug delivery.

scholarly journals ДОКЛІНІЧНЕ ДОСЛІДЖЕННЯ НАНОЧАСТИНОК ФЕРУМУ

2021 ◽  
pp. 17-24
Author(s):  
Y. S. Stravskyy ◽  
L. Ya. Fedoniuk ◽  
O. M. Yarema ◽  
E. І. Skyba ◽  
L. S. Reznichenko
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Candida Albicans ◽  
Salmonella Typhimurium ◽  
Proteus Mirabilis ◽  
Shigella Sonnei ◽  
Proteus Vulgaris

Вступ. Доклінічне вивчення лікарських препаратів – невід’ємна частина процесу створення лікарського засобу. Доклінічне дослідження є найбільш тривалим та відповідальним етапом розробки лікарського засобу, який вимагає особливих підходів до планування і забезпечення якості при плануванні вимірювальних експериментів, проведенні випробування та оцінки його результатів. Мета дослідження – визначити біобезпечність, гостру токсичність, протимікробну та фунгіцидну дії наночастинок Феруму. Методи дослідження. Біобезпечність синтезованої субстанції наночастинок у тестах in vitro визначали з використанням показників цитотоксичності, мутагенності, молекулярно-генетичного (показник генотоксичності), фізіологічного (стан мікрофлори шлунково-кишкового тракту людини) та біохімічних (ATФ-aзна і лактатдегідрогеназна активність) маркерів. Протимікробну дію нуль-валентного Феруму (Fe0NP) щодо тест-штамів мікроорганізмів визначали методом серійних розведень у бульйоні відповідно до Методичних вказівок 4.2.1890-04, 2004. Використовували такі тест-штами мікроорганізмів, як Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris, Proteus mirabilis, Candida albicans, із колекції Державного науково-контрольного інституту біотехнології і штамів мікро­організмів. Результати й обговорення. Синтезовані наночастинки є частинками Fe0NP. Взаємодія синтезованих наночастинок Феруму з тестовими еукаріотичними клітинами не призводила до появи первинних ДНК‑ушко­джень порівняно з впливом N-нітрозометилсечовини, яка є відомим генотоксикантом. Синтезовані наночастинки характеризувались як біобезпечні у тестах на мутагенність з використанням поліхроматофільних еритроцитів кісткового мозку тварин. Аналіз показав, що експериментальна субстанція Fe0NP у досліджуваному концентраційному діапазоні проявила помірну протимікробну активність у тестах in vitro відносно як грамнегативних (S. typhimurium, S. sonnei, P. aeruginosa, P. vulgaris, P. mirabilis), так і грампозитивних (S. aureus) мікроорганізмів. Однак гриби Candida albicans виявилися нечутливими до наночастинок Феруму в досліджуваних концентраціях. Висновки. Фізико-хімічна характеристика й оцінка критеріїв біобезпечності в тестах in vitro та in vivo свідчать про те, що синтезованим сферичним наночастинкам нуль-валентного Феруму властивий низький рівень потенційної небезпеки: виявлено відсутність генотоксичної, цитотоксичної, мутагенної дій, негативного впливу на ключові біохімічні параметри і загальний фізіологічний стан живого організму. Це дозволяє рекомендувати синтезовану субстанцію наночастинок Феруму для подальших досліджень з метою їх застосування як потенційної біологічно активної субстанції.

scholarly journals The 5’-NAD cap of RNAIII modulates toxin production in Staphylococcus aureus isolates

2019 ◽  
Author(s):  
Hector Gabriel Morales-Filloy ◽  
Yaqing Zhang ◽  
Gabriele Nübel ◽  
Shilpa Elizabeth George ◽  
Natalya Korn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Quorum Sensing ◽  
Biological Function ◽  
Toxin Production ◽  
Dual Function ◽  
Large Step ◽  
Opportunistic Human Pathogen ◽  
The Stability

1AbstractNicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5’-ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here we report the occurrence of several NAD-RNAs in the opportunistic human pathogen Staphylococcus aureus. Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium’s physiology, was found to be 5’-NAD-capped to a significant extent. NAD incorporation efficiency into RNAIII was found to depend in vivo on the −1 position of the P3 promoter. Reduction of RNAIII’s NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects to not seem to be due to changes in RNAIII’s secondary structure upon NAD attachment, as indicated by largely unaltered patterns in in vitro chemical probing experiments. Our study represents a large step towards establishing a biological function of the 5’-NAD cap, which for RNAIII in S. aureus is to modulate the expression of virulence factors.2ImportanceNumerous organisms, including bacteria, are endowed with a 5’-NAD cap in specific RNAs. While the presence of the 5’-NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5’-NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating bacterium’s virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

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