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Neddylation Alleviates Methicillin-Resistant Staphylococcus aureus Infection by Inducing Macrophage Reactive Oxygen Species Production.

ABSTRACT: Neddylation, a posttranslational modification in which NEDD8 is covalently attached to target proteins, has emerged as an endogenous regulator of innate immunity. However, the role of neddylation in methicillin-resistant Staphylococcus aureus (MRSA) infection remains unknown. In this study, we found that neddylation was activated after MRSA infection in vivo and in vitro. Inhibition of neddylation with MLN4924 promoted injury of liver and kidneys in C57BL/6 mice with MRSA bloodstream infection and increased mortality. Blockade of neddylation, either pharmacologically (MLN4924, DI591) or through the use of Uba3 small interfering RNA, inhibited Cullin3 neddylation and promoted Nrf2 accumulation, thus reducing reactive oxygen species (ROS) induction and bacterial killing ability in mouse peritoneal macrophages. In summary, our findings suggest that activation of neddylation in macrophages plays a critical protective role against MRSA infection by increasing ROS production, partially by signaling through the NEDD8-Cullin3-Nrf2-ROS axis. Furthermore, our results may provide a new non-antibiotic treatment strategy for MRSA infection through targeting of neddylation.

SUBMITTER: Xiu H

PROVIDER: S-EPMC8278275 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Antibiotic-resistant Staphylococcus aureus strains constitute a major public health concern worldwide and are responsible for both health care- and community-associated infections. Here, we establish a robust and easy-to-implement model of oral S. aureus infection using Drosophila melanogaster larvae that allowed us to follow the fate of S. aureus at the whole-organism level as well as the host immune responses. Our study demonstrates that S. aureus infection triggers H2O2 production by the host via the Duox enzyme, thereby promoting antimicrobial peptide production through activation of the Toll pathway. Staphylococcal catalase mediates H2O2 neutralization, which not only promotes S. aureus survival but also minimizes the host antimicrobial response, hence reducing bacterial clearance in vivo. We show that while catalase expression is regulated in vitro by the accessory gene regulatory system (Agr) and the general stress response regulator sigma B (SigB), it no longer depends on these two master regulators in vivo. Finally, we confirm the versatility of this model by demonstrating the colonization and host stimulation capabilities of S. aureus strains belonging to different sequence types (CC8 and CC5) as well as of two other bacterial pathogens, Salmonella enterica serovar Typhimurium and Shigella flexneri. Thus, the Drosophila larva can be a general model to follow in vivo the innate host immune responses triggered during infection by human pathogens. IMPORTANCE The pathogenicity of methicillin-resistant S. aureus (MRSA) strains relies on their ability to produce a wide variety of tightly regulated virulence factors. Current in vivo models to analyze host-pathogen interactions are limited and difficult to manipulate. Here, we have established a robust and reliable model of oral S. aureus infection using Drosophila melanogaster larvae. We show that S. aureus stimulates host immunity through the production of reactive oxygen species (ROS) and antimicrobial peptide (AMP) and that ROS potentialize AMP gene expression. S. aureus catalase plays a key role in this complex environment and acts in vivo independently from SigB and Agr control. We propose that fly larvae can provide a general model for studying the colonization capabilities of human pathogens.

Project description:Methicillin resistant Staphylococcus aureus (MRSA) infection is becoming refractory to existing antibiotic therapy owing to the inherent ability of S. aureus to develop rapid resistance and is considered a major threat to public health. We found that a natural isolate of Bacillus pumilus from the Columbia River Estuary produces a strong anti-MRSA compound, amicoumacin A. As amicoumacin A has been reported to exhibit anti-microbial, anti-inflammatory, and anti-ulcer activities, we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in the expression of genes involved in several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiholin-like product, which has been shown to be induced in response to a collapse of membrane potential. In order to gain further insight into the mechanism of action of amicoumacin A, a whole genome comparison of wild-type COL and amicoumacin A-resistant mutants isolated by serial passage method was carried out. Single point mutations resulting in codon substitutions were uncovered in several distinct genes: ksgA, RNA dimethyltranferase; fusA, elongation factor G; dnaG, primase, ; lacD, tagatose 1,6-bisphosphate aldolase, ; and SACOL0611, encoding a putative glycosyl transferase gene. Based on these results, a candidate approach was undertaken to recreate the same amino acid substitution individually in FusA and KsgA, each of which resulted in two-fold resistance towards amicoumacin A. The fusA gene is known as the site for fusidic acid- resistant mutations; however the codon substitutions in EF-G that cause amicoumacin A resistance and fusidic acid resistance occur in separate domains and do not bring about cross resistance. Taken together, these results suggest that amicoumacin A might cause perturbation of the cell membrane and lead to energy dissipation. Decreased rates of cellular metabolism including protein synthesis and DNA replication in resistant strains might allow cells to compensate for membrane dysfunction and thus increase cell survivability.

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Neddylation Alleviates Methicillin-Resistant Staphylococcus aureus Infection by Inducing Macrophage Reactive Oxygen Species Production.

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