Identifying potential therapeutic targets of methicillin-resistant Staphylococcus aureus through in vivo proteomic analysis

Abstract

Background: Detailed knowledge on protein repertoire of a pathogen during host infection is needed for both developing a better understanding of the pathogenesis and defining potential therapeutic targets. Such data, however, have been missing for Staphylococcus aureus, a major human pathogen.

Methods: We determined the surface proteome of methicillin-resistant S. aureus (MRSA) clone usa300 derived directly from murine systemic infectiON.

Results: The majority of the in vivo-expressed surface-associated proteins were lipoproteins involved in nutrient acquisition, especially uptake of metal ions. Enzyme-linked immunosorbent assay (ELISA) of convalescent human serum samples revealed that proteins that were highly produced during murine experimental infection were also produced during natural human infection. We found that among the 7 highly abundant lipoproteins only MntC, which is the manganese-binding protein of the MntABC system, was essential for MRSA virulence during murine systemic infection. Moreover, we show that MntA and MntB are equally important for MRSA virulence.

Conclusions: Besides providing experimental evidence that MntABC might be a potential therapeutic target for the development of antibiotics, our in vivo proteomics data will serve as a valuable basis for defining potential antigen combinations for multicomponent vaccines.

Keywords: MRSA; Staphylococcus aureus; bacterial pathogenesis; proteomics; vaccine and antibiotic development.

Figure 1.

Surface proteome of MRSA USA300 derived from in vivo infection and nutrient-limiting liquid culture in vitro. Relative abundance of surface-associated proteins is shown based on percentile of number of peptides detected for each class of protein relative to the total number of detected peptides. Proteins are grouped on predicted cellular location (red, sortase-anchored protein; blue, lipoprotein; green, integral membrane protein; yellow, secreted protein) and function (1, cell wall metabolism; 2, unknown function; 3, toxin; 4, protease; 5, host cell binding; 6, nutrient acquisition; 7, other functions). Abbreviations: dpi, days postinfection; MRSA, methicillin-resistant Staphylococcus aureus; RPMI, Roswell Park Memorial Institute media.

Figure 2.

ELISA analysis of human convalescent sera against surface-associated proteins highly produced during murine systemic infection. Serum samples from individual convalescent patients (n = 5) were tested against 9 recombinantly produced proteins. Proteins are indicated either by their names or by their accession numbers based on USA300_FPR3757 genome. BSA protein was used as a negative control. Medians of patient serum titers at A₄₅₀ = 1 are shown in horizontal bars. Abbreviations: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay.

Figure 3.

Inactivation of the gene encoding the MntC lipoprotein results in a severe attenuation of MRSA USA300 virulence in a murine systemic infection model. Kidney bacterial load of USA300 MRSA lipoprotein deletion mutants (deleted genes are shown in their names or in their locus numbers based on USA300_FPR3757 genome) 7 days postintravenous infection of 2 × 10⁶ CFU bacteria into A/J mice (n = 7). Data show the geometrical mean with 95% confidence intervals. Statistical analyses were performed by unpaired Mann–Whitney t tests. Dashed lines show the detection limit (83 CFU). Abbreviations: CFU, colony-forming units; MRSA, methicillin-resistant Staphylococcus aureus; WT, wild type.

Figure 4.

MntABC system is an essential virulence factor of MRSA during murine systemic infection. A, Operon organization of mntABC (top); schematic of the MntABC transport system (bottom). B, Kidney bacterial load of USA300 MRSA mntC_stop mutant and the mntC_{stop_repaired} strain 7 days post intravenous infection of 2 × 10⁶ CFU bacteria into A/J mice (n = 7). Data show the geometrical mean with 95% confidence intervals. Statistical analyses were performed by unpaired Mann–Whitney t tests. Dashed lines show the detection limit (83 CFU). C, Survival of A/J mice after intravenous infection of 5 × 10⁸ CFU USA300 MRSA wild-type (black line) and mntC_stop mutant (blue line) strains (n = 8). Statistical analysis was performed by the Mantel-Cox test. D, Kidney bacterial load of USA300 MRSA stop-codon insertion mutants in the mntABC operon 7 days post intravenous infection of 2 × 10⁶ CFU bacteria into A/J mice (n = 7). Data show the geometrical mean with 95% confidence intervals. Statistical analyses were performed by unpaired Mann–Whitney t tests. Dashed lines show the detection limit (83 CFU). Western blot analysis with anti-MntC antibodies shows that inactivation of mntA and mntB does not affect the expression of mntC (top panel). Abbreviations: CFU, colony-forming units; MRSA, methicillin-resistant Staphylococcus aureus; WT, wild type.