β-lactam Antibiotics Stimulate the Pathogenicity of Methicillin-resistant Staphylococcus aureus Via SarA-controlled Tandem Lipoprotein Expression

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of nosocomial infections worldwide. MRSA resists nearly all β-lactam antibiotics that have a bactericidal activity and a signal inducer effect. However, studies have yet to clarify whether the inducer effect of empirically used β-lactams stimulates MRSA pathogenicity in vivo. Here, we showed that a new cluster of tandem lipoprotein genes (tlpps) was upregulated in MRSA in response to the subinhibitory concentrations of β-lactam induction. The increased Tlpps significantly altered immune responses by macrophages with high IL-6 and TNFα levels. The deletion of the tlpps mutant (N315Δtlpps) significantly decreased the proinflammatory cytokine levels in vitro and in vivo. The bacterial loads of N315Δtlpps in the mouse kidney were also reduced compared with those of the wild type N315. The β-lactam-treated MRSA exacerbated cutaneous infections with increased lesion size, extended illness, and flake-like abscess-formation compared with those of the nontreatment. The β-lactam antibiotics that promoted the MRSA pathogenicity were SarA dependent, and the increasing expression of tlpps after β-lactam treatment was directly controlled by the global regulator SarA. Overall, our findings suggested that β-lactams should be used carefully because it might lead to a worse outcome of MRSA infection than inaction in the treatment. Author summary β-lactams are widely used in practice to treat infectious diseases, however, β-lactams worsening the outcome of a certain disease is poorly understood. In this study, we have identified a new cluster of tandem lipoprotein genes (tlpps) that is upregulated in the major clinically prevalent MRSA clones in response to the subinhibitory concentrations of β-lactams induction. The major highlight in this work is that β-lactams induce SarA expression, and then SarA directly binds to the tlpp cluster promoter region and upregulates the tlpp expression in MRSA. Moreover, the β-lactam stimulated Tlpps are important virulence factors that enhance MRSA pathogenicity. The deletion of the tlpps mutant significantly decreases the proinflammatory cytokine levels in vitro and in vivo. The β-lactam induced Tlpps enhance the host inflammatory responses by triggering the expression of IL-6 and TNFα, thereby promoting bacterial colonization and abscess formation. These data elucidate that β-lactams can worsen the outcome of MRSA infection through the induction of tlpps that are controlled by the global regulator SarA.

tandem, which is referred to as "tandem lipoproteins" (tlpps) or "lipoprotein-like" 107 (lpl) [15,18]. This tlpp cluster likely represents the paralogous genes that have 108 diverged after a duplication event in S. aureus [17]. MRSA USA300 belonging to the 109 clonal complex CC8 carries 15 (22%) hypothetical Tlpps. Of these Tlpps, 9 are 110 specific to the νSaα genomic island [15]. By comparison, N315 belonging to the 111 clonal complex CC5 carries 12 (21%) hypothetical Tlpps. Of these Tlpps,9 Lpl 112 proteins are specific to the νSaα genomic island (S1 Table). 113 Some staphylococcal Lpps can trigger host cell invasion, increase bacterial 114 pathogenicity, and contribute to the epidemic of CC8 and CC5 strains [19,20].   OXA-treated MRSA compared with the nontreatment one ( Fig 1B). 148 The protein band was excised from the SDS-PAGE gel and analyzed through 149 liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize the 150 β-lactam-induced proteins in the MRSA strains. The detected peptides matched with 151 68 proteins in the N315 proteome (S3 Table). Most known metabolic enzymes were 152 excluded, and three putative Tlpps, namely, Tlpp3 (SA2273), Tlpp2 (SA2274), and 153 Tlpp1(SA2275), encoded by a consecutive gene cluster were selected on the basis of 154 theoretical molecular weights for the analysis (Fig 2A and S1 Table). SA2273,  Table). This observation was consistent with the finding that  Reverse-transcription polymerase chain reaction (RT-PCR) was performed using 175 RNA extracted from MRSA N315 by specific primers to test whether tlpp1, tlpp2, 176 and tlpp3 were co-transcribed (Fig 2A and S5 Table). The comparison of the RT-PCR 7 177 results with the template of the genomic DNA or RNA only revealed that tlpp1, tlpp2, 178 and tlpp3 were co-transcribed from the tlpp1 promoter ( Fig 2B). We further examined 179 the influence of β-lactams on tlpp expression. Reverse-transcription quantitative PCR 180 (RT-qPCR) showed that the mRNA levels of tlpp1, tlpp2, and tlpp3 were upregulated 181 in N315 after treatment with the subinhibitory concentrations of OXA ( Fig 2C).

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Western blot analysis demonstrated that the protein levels of Tlpps in both N315 total 183 cell lysates (Fig 2D and 2E) and the culture supernatant (Fig 2F and 2G) increased in 184 a dose-dependent manner after OXA treatment was administered. Tlpp expression   N315Δtlpps for 5 days (S6 Table), and bacterial colonization was tracked through an 234 animal imaging system. The fluorescence intensity of the GFP in the murine organs 235 (i.e., heart, lung, liver, spleen, and kidney) was measured, and the results indicated 236 that the radiant efficiency in the kidneys of the mice injected with N315 was 9 237 significantly higher than those infected with N315Δtlpps (Fig 4C and 4D). Consistent 238 with the radiant efficiency, the bacterial loads in the kidneys of the N315-infected 239 mice were also significantly higher than that of the N315Δtlpps-infected ones ( To investigate the effect of SarA on β-lactam-stimulated Tlpp expression, we 301 constructed a reporter vector (pOS1-tlpps P ) containing the tlpp promoter-controlled 302 lacZ gene (S6 Table) and performed β-galactosidase assay by transforming 303 pOS1-tlpps P into the MRSA strains N315 and N315ΔsarA. The results revealed that 304 the β-galactosidase activity was significantly lower in the sarA mutant than that in 305 N315. Moreover, the β-galactosidase activity presented no significant change in the 306 sarA mutant after OXA treatment compared with the untreated N315ΔsarA ( Fig 7A).  widely distributed among the major prevalent MRSA clones (Fig 1B and S4 Table).

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In addition to antimicrobial activity, signal induction may be implemented by the  (Fig 3A and 3B).  were the most upregulated regulators in MRSA N315 after OXA treatment (Fig 6A).

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The deletion of SarA (N315ΔsarA) failed to upregulate tlpps even under OXA 386 treatment (Fig 7A and 7B), indicating that the β-lactam-induced Tlpp expression in 387 MRSA was SarA controlled. EMSA data revealed that the regulation of SarA on Tlpp 388 expression was direct (Fig 7F). However, further investigations should be performed 389 to clarify how β-lactams trigger the SarA expression.      Table. 489 490

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The tlpp cluster marker-less deletion mutant was constructed using homologous  To construct the tlpps complementary strain, the tlpp cluster containing its 503 potential promoter region was amplified by pLI-tlpps fwd/pLI-tlpps rev primer pairs, 18 504 cloned into the expression plasmid pLI50 [40]. Then, the correctly constructed 505 plasmid pLI-tlpps was electroporated into RN4220 and then N315Δtlpps to generate 506 N315Δtlpp/pLI-tlpps strain. Similar strategy was used to construct 507 N315ΔsarA/pLI-sarA. The empty pLI50 plasmid transformed N315Δtlpps and 508 N315ΔsarA strains served the controls. All primers used are listed in S5 Table.  LacZ activity was normalized to the cell density of OD600, and the relative activity 559 was calculated by setting the LacZ activity from the N315 to 100%. The assay was 560 repeated at least three times.

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Electrophoretic mobility shift assays (EMSA) 563 The predicted tlpp cluster promoter, an AT-rich motif fragment (56 bp), was 20 564 synthesized using the primer pairs (EMSA-tlpps P fwd/EMSA-tlpps P rev) as described 565 [45]. The corresponding mutated GC-rich motif fragment was also synthesized by 566 primer pairs EMSA-tlpps PM fwd/EMSA-tlpps PM rev and served as the control. Ten   Table . 575 576 Statistical analysis 577 Statistical analysis was carried out using GraphPad Prism 6.0. Unpaired two-tailed 578 student's t-test, analysis of variance (ANOVA) and Mann-Whitney test were used 579 appropriately to compare the difference between groups. Each experiment was carried 580 out at least three times. Results were presented as mean ± standard deviations (S.D.), 581 and a P value less than 0.05 was considered statistically significant. * P < 0.05, ** P 582 < 0.01, *** P < 0.001, and ns represented no significance.  μg/ml (S2 Table) because of the heterogeneous β-lactam-resistant phenotype of 654 MRSA [46]. Therefore, the subinhibitory concentrations of OXA, 2 μg/ml, was used 655 unless specifically stated in this study.

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(TIF) 657 S10 Fig. Full Western blot data. The full-length blots for all Western blot pictures.

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The black boxes represented the depicted parts of the blot.