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Exploring the susceptome of methicillin-resistant Staphylococcus aureus to β-Lactams using a multi-omics approach

ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant challenge to public health due to its evolving resistance to antibiotics. In addressing this concern, we employed a multi-omics approach, integrating proteomics and Transposon Directed Insertion-site Sequencing (TraDIS), to comprehensively investigate adaptive response of MRSA to β-lactam antibiotics and identify key genetic determinants influencing susceptibility. Proteomic analysis revealed significant shifts in the protein spectrum under sub-inhibitory concentrations of oxacillin and cefazolin, characterized by a stringent response. This included the upregulation of amino acid metabolism and oligopeptide transport pathways, along with the downregulation of arginine biosynthesis. Concurrent TraDIS screening identified 50 genes whose inactivation conferred a fitness advantage in the presence of both β-lactams, implicating processes such as nucleotide second messenger signaling, DNA repair, and histidine transport. Validation of selected mutants (relA::Tn, mutY::Tn, nsaR::Tn, aroC::Tn, SAUSA300_0432::Tn, SAUSA300_0846::Tn, SAUSA300_0595::Tn) confirmed these findings, demonstrating improved fitness in the presence of β-lactams. Integrative analysis of proteomic and TraDIS datasets highlighted the implication of the stringent response in mediating MRSA adaptation to β-lactams. These findings establish the 'susceptome' concept, a collection of genes that maintain or potentiate MRSA's vulnerability to β-lactams, providing new targets for therapeutic intervention and strategies to counteract resistance evolution.

INSTRUMENT(S):

ORGANISM(S): Staphylococcus Aureus

SUBMITTER: Nader Abdelmalek

LAB HEAD: Bianca Paglietti

PROVIDER: PXD062516 | Pride | 2025-12-12

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	CTvsCef_USA300DB.pdResult	Other
	J-JC_CefazolinUSA300DB_Proteins.txt	Txt
	J-JO_OxacillinUSA300DB.txt	Txt
	J-JO_USA300DB_Minora.msf	Msf
	J-JO_USA300DB_area-norm.pdResult	Other

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Project description:Subinhibitory concentrations of the neuroleptic drug thioridazine (TDZ) are well-known to enhance the killing of methicillin-resistant S. aureus (MRSA) by Î²-lactam antibiotics, however, the mechanism underlying the synergy between TDZ and Î²-lactams is not fully understood. In the present study we have examined the effect of a subinhibitory concentration of TDZ on antimicrobial resistance, the global transcriptome, and the cell wall composition of MRSA USA300. We show that TDZ is able to sensitize the bacteria to several classes of antimicrobials targeting the late stages of peptidoglycan synthesis. Furthermore, our microarray analysis demonstrates that TDZ modulates the expression of genes encoding membrane and surface proteins, transporters, and enzymes involved in amino acid biosynthesis. Interestingly, resemblance between the transcriptional profile of TDZ treatment and the transcriptomic response of S. aureus to known inhibitors of cell wall synthesis suggests that TDZ disturbs peptidoglycan biosynthesis at a stage that precedes transpeptidation. In support of this notion, dramatic changes in the muropeptide profile of USA300 were observed following growth in the presence of TDZ, indicating that TDZ can interfere with the formation of the pentaglycine branches. Strikingly, the addition of glycine to the growth medium relieved the effect of TDZ on the muropeptide profile. Furthermore, exogenous glycine offered a modest protective effect against TDZ-induced Î²-lactam sensitivity. We propose that TDZ exposure leads to a shortage of intracellular amino acids, including glycine, which is required for the production of normal peptidoglycan precursors with pentaglycine branches, the correct substrate of S. aureus penicillin-binding proteins. Collectively, this work demonstrates that TDZ has a major impact on the cell wall biosynthesis pathway in S. aureus and provides new insights into how MRSA may be sensitized towards Î²-lactam antibiotics. Staphylococcus aureus USA300 was grown to early exponential phase and treated with TDZ (16 Âµg/ml) alone or in combination with DCX (0.125 Âµg/ml) for 30 min. Changes in global gene expression were analyzed using the untreated culture as control. Hybridizations were performed in triplicate using RNA isolated from independent cultures.

Project description:Microglia are the resident mononuclear phagocytes of the CNS parenchyma and represent an initial line of defense against invading microorganisms. Microglia utilize Toll-like receptors (TLRs) for pathogen recognition and TLR2 specifically senses conserved motifs of gram-positive bacteria including lipoproteins, lipoteichoic acids, and peptidoglycan (PGN) leading to cytokine/chemokine production. Interestingly, primary microglia derived from TLR2 knockout (KO) mice over-expressed numerous IL-12 family members, including IL-12p40, IL-12p70, and IL-27 in response to intact S. aureus, but not the less structurally complex TLR2 ligands Pam3CSK4 or PGN. The ability of intact bacteria to augment IL-12 family member expression was specific for gram-positive organisms, since numerous gram-negative strains were unable to elicit exaggerated responses in TLR2 KO microglia. Inhibition of SYK or IRAK4 signaling did not impact heightened IL-12 family member production in S. aureus-treated TLR2 KO microglia, whereas PI3K, MAPK, and JNK inhibitors were all capable of restoring exaggerated cytokine expression to wild type levels. Additionally, elevated IL-12 production in TLR2 KO microglia was ablated by a TLR9 antagonist, suggesting that TLR9 drives IL-12 family member production following exposure to intact bacteria that remains unchecked in the absence of TLR2 signaling. Collectively, these findings indicate crosstalk between TLR2 and TLR9 pathways to regulate IL-12 family member production by microglia. The summation of TLR signals must be tightly controlled to ensure the timely cessation and/or fine tuning of cytokine signaling to avoid nonspecific bystander damage due to sustained IL-12 release. TLR2 KO mice were backcrossed with C57BL/6 animals for a minimum of eight generations prior to use in these studies. Age- and sex-matched C57BL/6 mice were used as wild type (WT) controls. Primary mixed glial cultures were prepared from the cerebral corticies of neonatal mice (2-4 days of age) and microglia were harvested using a differential shaking technique with a purity of >98%. A USA300 community-acquired methicillin-resistant S. aureus (CA-MRSA) clinical isolate recovered from a patient with a fatal brain abscess was used to stimulate the microglia isolates. Bacterial strains were heat-inactivated and used to stimulate microglia at 107 colony forming units (cfu)/well for 6 and 12 hours time points. Three replicates of each mouse type (WT, TLR2 KO) at both time points 6 and 12 hours were used for the microarray experiments. Data was only usable for 2 replicates of the KO-12 hr group.

Project description:The precise mechanism and effects of antibiotics in host gene expression and immunomodulation in MRSA infection is unknown. Using a well characterized Methicillin Resistant Staphylococcus aureus (MRSA) isolate USA300 in a murine model of infection, we determined that linezolid and vancomycin induced differential production of bacterial toxins and host cytokines, differences in host gene expression, and differences in immunomodulators during MRSA bloodstream infection. A total of 35 A/J mice, categorized into seven groups (no infection; no infection with linezolid; no infection with vancomycin; 2 hour post-infection (hpi) S. aureus; 24 hpi S. aureus; 24 hpi S. aureus with linezolid; and 24 hpi S. aureus with vancomycin), were used in this study. Mice were injected with USA300 (6 x 106 CFU/g via i.p. route), then intravenously treated with linezolid (25 mg/kg) or vancomycin (25 mg/kg) at 2 hpi. Control and S. aureus infected mice were euthanized at each time point (2 h or 24h) following injection. Whole blood RNA was used for microarray; three cytokines and two S. aureus toxins [PantonValentine Leukocidin (PVL) and alpha hemolysin] were quantified in mouse serum by ELISA. S. aureus CFUs were significantly reduced in blood and kidney after linezolid or vancomycin treatment in S. aureus-infected mice. In vivo IL-1M-NM-2 in mouse serum was significantly reduced in both linezolid (p=0.001) and vancomycin (p=0.006) treated mice compared to untreated ones. IL-6 was significantly reduced only in linezolid treated (p<0.001) but not in vancomycin treated mice. However, another proinflammatory cytokine, TNF-M-NM-1, did not exhibit altered levels in either linezolid or vancomycin treated mice (p=0.3 and p=0.51 respectively). In vivo level of bacterial toxin, Panton-Valentine leukocidin, in mouse serum was significantly reduced only in linezolid treated mice (p=0.02) but not in vancomycin treated mice. There was no significant effect of either treatment in in vivo level of alpha hemolysin production. Unsupervised hierarchical clustering using the gene expression data from 35 microarrays revealed distinct clustering based on infection status and treatment group. Study of the antibiotic-specific difference in gene expression identified the number of genes uniquely expressed in response to S. aureus infection, infection with linezolid treatment, and infection with vancomycin treatment. Pathway associations study for the differentially expressed genes in each comparison group (Control vs. 24 h S. aureus infection, 24 h S. aureus infection vs. 24 h S. aureus linezolid, and 24 h S. aureus infection vs. 24 h S. aureus vancomycin) in mice using Kyoto Encyclopedia of Genes and Genomes (KEGG) identified toll-like receptor signaling pathway to be common to every comparison groups studied. Glycerolipid metabolism pathway was uniquely associated only with linezolid treatment comparison group. The findings of this study provide the evidence that protein synthesis inhibitor like linezolid does a better job in treating MRSA sepsis compared to cell wall acting antibiotics like vancomycin. To identify differences in host gene expression in a murine sepsis model treated with a) linezolid and b) vancomycin, we used whole blood gene expression (RNA) signatures from A/J inbred mice infected with USA 300 MRSA to evaluate differences in host gene expression among mice treated with linezolid and vancomycin. We used 5 RNA samples from MRSA-infected, linezolid- or vancomycin-treated mice. A total of 7 experimental groups have been employed: 1) Uninfected control group: (negative controls). 2) Uninfected, linezolid-treated group: Uninfected, linezolid-treated mice. 3) Uninfected vancomycin-treated group: Uninfected, vancomycin-treated mice. 4) Infected control group (positive control 2 h) MRSA-infected, untreated mice. 5) Infected control group (positive control 24 h): MRSA-infected, untreated mice. 6) Infected linezolid group: MRSA-infected, linezolid-treated mice. 7) Infected vancomycin group: MRSA-infected, vancomycin-treated mice.

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Exploring the susceptome of methicillin-resistant Staphylococcus aureus to β-Lactams using a multi-omics approach

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