<i>Staphylococcus aureus</i>ST228 and ST239 as models for expression studies of diverse markers during osteoblast infection and persistence

Bongiorno, Dafne; Musso, Nicolò; Caruso, Giuseppe; Lazzaro, Lorenzo; Caraci, Filippo; Stefani, Stefania; Campanile, Floriana

doi:10.1002/mbo3.1178

Cited by 7 publications

(12 citation statements)

References 49 publications

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“…The evolution of reduced Agr functionality (and thus cytotoxicity) in hospital-acquired ST239 and ST22 isolates has already been reported by our group and others and is confirmed by the InToxSa outputs ( Baines et al, 2015 ; Collins et al, 2008 ; Giulieri et al, 2018 ; Laabei et al, 2021 ; Li et al, 2016 ). Consistent with their reduced cytotoxicity and with our hypothesis of inverse correlation between toxicity and intracellular replication, ST239 isolates caused higher degrees of bacterial persistence in infected animal models ( Baines et al, 2015 ; Li et al, 2016 ) and showed increased intracellular persistence in osteoblasts ( Bongiorno et al, 2021 ). Within the limits of our experimental settings, the relatively lower cytotoxicity of ST239 and ST22 isolates indicates that the amplitude of this phenotype should probably be considered within a genetic lineage.…”

Section: Discussionsupporting

confidence: 81%

A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence

Hachani

Giulieri

Guérillot

et al. 2023

eLife

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Staphylococcus aureus infections are associated with high mortality rates. Often considered an extracellular pathogen, S. aureus can persist and replicate within host cells, evading immune responses, and causing host cell death. Classical methods for assessing S. aureus cytotoxicity are limited by testing culture supernatants and endpoint measurements that do not capture the phenotypic diversity of intracellular bacteria. Using a well-established epithelial cell line model, we have developed a platform called InToxSa (intracellular toxicity of S. aureus) to quantify intracellular cytotoxic S. aureus phenotypes. Studying a panel of 387 S. aureus bacteraemia isolates, and combined with comparative, statistical, and functional genomics, our platform identified mutations in S. aureus clinical isolates that reduced bacterial cytotoxicity and promoted intracellular persistence. In addition to numerous convergent mutations in the Agr quorum sensing system, our approach detected mutations in other loci that also impacted cytotoxicity and intracellular persistence. We discovered that clinical mutations in ausA, encoding the aureusimine non-ribosomal peptide synthetase, reduced S. aureus cytotoxicity, and increased intracellular persistence. InToxSa is a versatile, high-throughput cell-based phenomics platform and we showcase its utility by identifying clinically relevant S. aureus pathoadaptive mutations that promote intracellular residency.

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Section: Discussionsupporting

confidence: 81%

A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence

Hachani

Giulieri

Guérillot

et al. 2023

eLife

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“…RNA extraction was performed using the RNeasy Mini Kit (Cat. No 74104, Qiagen, Hilden, Germany) following the manufacturer's instructions, with some modifications as previously described [17] RNA quality was tested with a Qubit® 3.0 Fluorometer (Cat. No Q33216, Life Technologies, Thermo Fisher Scientific, Waltham, Massachusetts, USA) using the Qubit RNA HS Assay Kit (250 pg/µl and 100 ng/µl).…”

Section: Rna Extractionmentioning

confidence: 99%

“…Furthermore, the agrA, sdrE, psmA, hla, hld, uhpt and SigB genes were amplified using the primers previously reported [17]. RNA was normalized at 50 ng/µl using the QuantiTect Reverse Transcription Kit (Cat.…”

Section: Primer Designmentioning

confidence: 99%

“…Based on our knowledge, intracellular survival of S. aureus varies depending on the set of virulence factors produced (e.g., PVL toxin, alpha toxin, etc) as well as its genetic background. This finding was already assessed through internalization assays in human MG-63 osteoblastlike cells, which revealed differences in internalization and intracellular persistence of S. aureus based on its sequence type (ST) [17]. The complications related to the treatment of persistent and chronic infections has led to the development of novel therapeutic strategies.…”

Section: Introductionmentioning

confidence: 99%

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Phagosomal Escape, Intracellular Survival and Induction of Small Colony Variants of <em>Staphylococcus aureus</em> USA300 in RAW 264.7 Murine Macrophages

Bivona¹,

Bonomo²,

Bonacci³

et al. 2023

Preprint

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Phagosomal escape and intracellular survival, often accompanied by Small Colony Variants (SCVs) formation, are typical features of infections caused by S. aureus. The survival in macro-phages favours S. aureus dissemination and complicates treatment. RAW 264.7 murine macro-phages infected with S. aureus USA300 and treated with erythromycin and 20mM carnosine, alone and in combination, were used as experimental model. SCVs were isolated from all treat-ment conditions, but only those undergoing the pressure of combined erythromycin and carnosine for 48 hours were stable for at least six passages on blood agar. Nucleic acid extraction was car-ried out for S. aureus USA300 wild-type and stable SCVs. Whole Genome Sequencing (WGS) was performed using Illumina DNA Prep and Illumina MiSeq, and quantitative reverse transcription PCR was performed. WGS analysis did not yield mutations pointing to differences between S. au-reus USA300 and stable SCVs, therefore the focus was shifted to evaluating gene expression vari-ations. Genes such as zur, mntR, uhpt, fur, sdrE were shown to be significantly up-regulated in SCVs compared to S. aureus USA300 wild-type, suggesting a global change that allows adapta-tion to intracellular persistence, including protection from inflammatory response and evasion of the immune system.

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“…Although different studies have described that S. aureus can survive for long periods within osteoblasts (Kalinka et al, 2014;Ji et al, 2020;Bongiorno et al, 2021;Gunn et al, 2021;Marro et al, 2021), the mechanisms that may explain the persistence within bone cells are still unknown. In addition to osteoblasts, several studies have revealed osteocytes as possible niche for bacteria within bone tissue (De Mesy Bentley et al, 2018;Yang et al, 2018;Krauss et al, 2019;Masters et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms

et al. 2022

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Chronic staphylococcal osteomyelitis can persist for long time periods causing bone destruction. The ability of Staphylococcus aureus to develop chronic infections is linked to its capacity to invade and replicate within osteoblasts and osteocytes and to switch to a dormant phenotype called small colony variants. Recently, osteocytes were described as a main reservoir for this pathogen in bone tissue. However, the mechanisms involved in the persistence of S. aureus within these cells are still unknown. Here, we investigated the interaction between S. aureus and osteoblasts or osteocytes during infection. While osteoblasts are able to induce a strong antimicrobial response and eliminate intracellular S. aureus, osteocytes trigger signals to recruit immune cells and enhance inflammation but fail an efficient antimicrobial activity to clear the bacterial infection. Moreover, we found that extracellular signals from osteocytes enhance intracellular bacterial clearance by osteoblasts. Even though both cell types express Toll-like receptor (TLR) 2, the main TLR responsible for S. aureus detection, only osteoblasts were able to increase TLR2 expression after infection. Additionally, proteomic analysis indicates that reduced intracellular bacterial killing activity in osteocytes is related to low antimicrobial peptide expression. Nevertheless, high levels of lipid mediators and cytokines were secreted by osteocytes, suggesting that they can contribute to inflammation. Taken together, our results demonstrate that osteocytes contribute to severe inflammation observed in osteomyelitis and represent the main niche for S. aureus persistence due to their poor capacity for intracellular antimicrobial response.

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Staphylococcus aureusST228 and ST239 as models for expression studies of diverse markers during osteoblast infection and persistence

Abstract: This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Cited by 7 publications

References 49 publications

A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence

A high-throughput cytotoxicity screening platform reveals agr-independent mutations in bacteraemia-associated Staphylococcus aureus that promote intracellular persistence

Phagosomal Escape, Intracellular Survival and Induction of Small Colony Variants of <em>Staphylococcus aureus</em> USA300 in RAW 264.7 Murine Macrophages

Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms

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