SpoVG modulates cell aggregation by regulating sasC expression and eDNA 1 release in Staphylococcus aureus

Biofilm formation is involved in numerous Staphylococcus aureus infections such as 11 endocarditis, septic arthritis, osteomyelitis, and infections on in-dwelling medical 12 devices. In these diseases, S. aureus forms biofilms as cell aggregates interspersed in 13 host matrix material. Here, we have observed that cell aggregation was significantly 14 higher in the isogenic spoVG -deletion strain compared with that of the wild-type strain. 15 Reverse transcription-quantitative PCR data indicated that SpoVG could repress the 16 expression of sasC , which codes for S . aureus surface protein C and is involved in cell 17 aggregation and biofilm accumulation. Electromagnetic mobility shift assay 18 demonstrated that SpoVG could specifically bind to the promoter region of sasC , 19 indicating that SpoVG is a negative regulator and directly represses the expression of 20 sasC . In addition, deletion of the SasC aggregation domain in the spoVG -deletion strain 21 indicated that high level expression of sasC could be the underlying cause of 22 significantly increased cell aggregation formation. Our previous study has shown that 23 SpoVG is involved in oxacillin resistance of methicillin-resistant S. aureus by 24 regulating the expression of genes involved in cell wall synthesis and degradation. In 25 this study, we also have found that SpoVG can negatively modulate the S. aureus drug 26 tolerance under high concentration of oxacillin treatment. These findings can broaden 27 our understanding of the regulation of biofilm formation and drug tolerance in S. aureus . This study has revealed that SpoVG can modulate cell aggregation by repressing sasC 31 expression and eDNA release. Furthermore, we have demonstrated the potential linkage 32 between cell aggregation and antibiotic resistance. Our findings provide novel insights 33 into the regulatory mechanisms of SpoVG involved in cell aggregation, biofilm 34 development and formation in Staphylococcus aureus.

INTRODUCTION structure development and surface coverage (5). A previous study has pointed to the 53 advantage of cell aggregations over single cells during biofilm formation (6). S. aureus 54 cell aggregation is a biological process through which cells bind to matrix proteins and 55 form stable clumps to evade host defenses and to adapt to antibiotic stress. In aggregate 56 communities, S. aureus cells adjust the distribution of their adhesins and surface 57 proteins to promote their tolerance to hazardous environments (7,8). Biofilm 58 development and formation can be modulated by various regulatory factors such as 59 Sigma B (9), Agr system (9), SaeRS (10, 11), SarA (10), and MgrA (12, 13), but the 60 regulatory mechanisms of cell aggregation remain largely unknown. 61 In S. aureus, SpoVG is a global transcriptional regulator and binds to the DNA region 62 that contains a characteristic TAATTT/A motif (14). SpoVG can modulate the 63 production of capsule, extracellular nuclease, protease, lipase (15)(16)(17), and emergence 64 of methicillin-and glycopeptide-resistance of methicillin-resistant S. aureus (MRSA) 65 and vancomycin-intermediate S. aureus (VISA) (15,18). 66 In this study, we found that cell aggregation was significantly increased in the S. aureus 67 spoVG-deletion strain compared with that of the wild-type (WT) strain. In addition, RT-68 qPCR data identified a potential target gene sasC. By introducing the spoVG sasC 69 double mutant, we demonstrated that SpoVG could modulate cell aggregation by  The spoVG-deletion strain forms stronger cell aggregation. 75 During the growth of S. aureus, we found a significant difference in bacterial behavior 76 between the WT and spoVG-deletion strains. The spoVG-deletion strain exhibited cell 77 clumps after grown for 3, 6, 9, 12 hours in transparent glass tubes compared with the 78 WT strain, and the alteration could be reversed by the spoVG complementation ( and spoVG-complemented strains (Fig. 1C). In addition, a fluorescence microscopy 86 was employed to determine the morphological feature of the spoVG-deletion strain with 87 the fluorescent shuttle plasmid pALC. As a result, fairly apparent cell clusters were 88 formed in the spoVGdeletion strain after growth overnight (Fig. 1D). These data 89 indicate that SpoVG plays a significant role in the regulation of cell aggregation.

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Cell aggregation of the spoVG-deletion strain is protease-sensitive 91 To analyze the components of cell aggregation formed in the spoVG-deletion strain, we 92 added proteinase K and trypsin into the cell aggregation culture, and PBS was added as 93 a control treatment. Cell clusters were dissolved after digested with proteinase K and 94 trypsin ( Fig. 2A-B), suggesting that the cell clusters formed in the spoVG-deletion 95 strain may involve the expression variation of bacterial surface proteins. showed that the transcriptional levels of 12 genes were altered in the spoVG-deletion 104 strain, including 3 up-regulated genes ebhB, isdA and sasC, and 9 down-regulated genes 105 clfB, sdrC, sraP, sasG, spa, sdrE, emp, eap and coa. Among these genes, the mRNA 106 levels of ebhB and sasC were significantly increased in the spoVG-deletion strain  resistance compared with that of the spoVG-deletion strain ( Fig. 7A-B). 172 We also tested cell survival of the WT, spoVG-deletion, spoVG-complemented, sasC 173 mutant, and spoVG sasC double mutant strains in MH broth exposed to 6.4 mg/ml of 174 oxacillin (representing approximately 100×MIC for the WT strain) for 24 or 48 hours.

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The spoVG-deletion strain exhibited significantly increased drug tolerance after treated   Furthermore, our data have indicated that SpoVG is involved in transcriptional 199 regulation of ebhB, and the underlying mechanism requires to be further studied. SpoVG in biofilm formation needs to be further studied. We have also revealed the 208 potential linkage between cell aggregation and antibiotic resistance, and the exact 209 mechanism needs to be further investigated (Fig. 8A-B). aureus RN4220 as the initial recipient and then S. aureus strain N315 by electroporation.

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The media were solidified with 1.5% (w/v) agar when needed.

Construction of the sasC single mutation and spoVG sasC double mutation 223
To obtain a single mutant of the sasC gene and spoVG sasC double mutation, the 224 plasmid pBTs and a protocol was used as described previously (22). Briefly, DNA 225 fragments corresponding to the upstream and downstream regions of sasC aggregation 226 domain were amplified by PCR, using S. aureus strain N315 genomic DNA as template.

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The PCR products were ligated by overlap PCR to form an up-down fragment, which   Table 2. All the RT-qPCR assays 259 were repeated at least three times.

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To construct the reporter plasmid pOSsasC, the DNA fragment containing the sasC 262 promoter region was amplified from S. aureus strain N315 genomic DNA using primers 263 listed in Table 2. The fragment was digested with BamHI and EcoRI and cloned into 264 the shuttle vector pOS1. The reporter plasmid was first transformed into S. aureus 265 RN4220 for modification, and then the WT and spoVG-deletion strains. The β-galactosidase activity analysis was performed as previously described (24). For The biotin-labeled DNA fragment, psasC containing sasC promoter region was 281 amplified from S. aureus strain N315 genomic DNA using primers listed in Table 2.

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The biotin-labeled psasC was incubated at 25°C for 30 minutes with various amounts Purification and detection of eDNA was performed as previously described (25). Mueller-Hinton agar with 2% NaCl containing increasing concentrations of oxacillin.

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The colony-forming units (CFU) were determined after overnight incubation at 37°C.