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CcpA Regulates Staphylococcus aureus Biofilm Formation through Direct Repression of Staphylokinase Expression.

ABSTRACT: Staphylococcus aureus represents a notorious opportunistic pathogen causing various infections in biofilm nature, imposing remarkable therapeutic challenges worldwide. The catabolite control protein A (CcpA), a major regulator of carbon catabolite repression (CCR), has been recognized to modulate S. aureus biofilm formation, while the underlying mechanism remains to be fully elucidated. In this study, the reduced biofilm was firstly determined in the ccpA deletion mutant of S. aureus clinical isolate XN108 using both crystal violet staining and confocal laser scanning microscopy. RNA-seq analysis suggested that sak-encoding staphylokinase (Sak) was significantly upregulated in the mutant ∆ccpA, which was further confirmed by RT-qPCR. Consistently, the induced Sak production correlated the elevated promoter activity of sak and increased secretion in the supernatants, as demonstrated by Psak-lacZ reporter fusion expression and chromogenic detection, respectively. Notably, electrophoretic mobility shift assays showed that purified recombinant protein CcpA binds directly to the promoter region of sak, suggesting the direct negative control of sak expression by CcpA. Double isogenic deletion of ccpA and sak restored biofilm formation for mutant ∆ccpA, which could be diminished by trans-complemented sak. Furthermore, the exogenous addition of recombinant Sak inhibited biofilm formation for XN108 in a dose-dependent manner. Together, this study delineates a novel model of CcpA-controlled S. aureus biofilm through direct inhibition of sak expression, highlighting the multifaceted roles and multiple networks regulated by CcpA.

SUBMITTER: Zheng M

PROVIDER: S-EPMC9598941 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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CcpA Regulates Staphylococcus aureus Biofilm Formation through Direct Repression of Staphylokinase Expression.

Zheng Mingxia M Zhu Keting K Peng Huagang H Shang Weilong W Zhao Yan Y Lu Shuguang S Rao Xiancai X Li Ming M Zhou Renjie R Li Gang G

Antibiotics (Basel, Switzerland) 20221017 10

Staphylococcus aureus represents a notorious opportunistic pathogen causing various infections in biofilm nature, imposing remarkable therapeutic challenges worldwide. The catabolite control protein A (CcpA), a major regulator of carbon catabolite repression (CCR), has been recognized to modulate S. aureus biofilm formation, while the underlying mechanism remains to be fully elucidated. In this study, the reduced biofilm was firstly determined in the ccpA deletion mutant of ...[more]

PMID: 36290085

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Project description:Staphylococcus aureus is a medically important pathogen with high metabolic versatility allowing it to infect various niches within a host. S. aureus utilizes two major transcriptional regulators, namely, CodY and CcpA, to remodel metabolic and virulence gene expression in response to changing environmental conditions. Previous studies revealed that inactivation of either codY or ccpA has a pronounced impact on different aspects of staphylococcal physiology and pathogenesis. To determine the contribution and interplay of these two regulators in modulating central metabolism, virulence, and biofilm development, we constructed and characterized the codY ccpA double mutant in S. aureus UAMS-1. In line with previous studies, we found that CcpA and CodY control the cellular metabolic status by altering carbon flux through the central and overflow metabolic pathways. Our results demonstrate that ccpA inactivation impairs biofilm formation and decreases incorporation of extracellular DNA (eDNA) into the biofilm matrix, whereas disrupting codY resulted in a robust structured biofilm tethered together with eDNA and polysaccharide intercellular adhesin (PIA). Interestingly, inactivation of both codY and ccpA decreases biofilm biomass and reduces eDNA release in the double mutant. Compared with the inactivation of codY, the codY ccpA mutant did not overexpress toxins but maintained overexpression of amino acid metabolism pathways. Furthermore, the codY ccpA mutant produced large amounts of PIA, in contrast to the wild-type strain and ccpA mutant. Combined, the results of this study suggest that the coordinated action of CodY and CcpA modulate central metabolism, virulence gene expression, and biofilm-associated genes to optimize growth on preferred carbon sources until starvation sets in. IMPORTANCE Staphylococcus aureus is a leading cause of biofilm-associated infections, including infective endocarditis, worldwide. A greater understanding of metabolic forces driving biofilm formation in S. aureus is essential for the identification of novel therapeutic targets and for the development of new strategies to combat this medically important pathogen. This study characterizes the interplay and regulation of central metabolism and biofilm development by two global transcriptional regulators, CodY and CcpA. We found that the lack of CcpA and/or CodY have different impacts on intracellular metabolic status leading to a formation of morphologically altered biofilms. Overall, the results of this study provide new insights into our understanding of metabolism-mediated regulation of biofilm development in S. aureus.

Project description:Staphylococcus aureus chronically colonizes up to 30% of the human population on the skin or mucous membranes, including the nasal tract or vaginal canal. While colonization is often benign, this bacterium also has the capability to cause serious infections. Menstrual toxic shock syndrome (mTSS) is a serious toxinosis associated with improper use of tampons, which can induce an environment that is favorable to the production of the superantigen known as toxic shock syndrome toxin-1 (TSST-1). To better understand environmental signaling that influences TSST-1 production, we analyzed expression in the prototype mTSS strain S. aureus MN8. Using transcriptional and protein-based analysis in two niche-related media, we observed that TSST-1 expression was significantly higher in synthetic nasal medium (SNM) than in vaginally defined medium (VDM). One major divergence in medium composition was high glucose concentration in VDM. The glucose-dependent virulence regulator gene ccpA was deleted in MN8, and, compared with wild-type MN8, we observed increased TSST-1 expression in the ΔccpA mutant when grown in VDM, suggesting that TSST-1 is repressed by catabolite control protein A (CcpA) in the vaginal environment. We were able to relieve CcpA-mediated repression by modifying the glucose level in vaginal conditions, confirming that changes in nutritional conditions contribute to the overexpression of TSST-1 that can lead to mTSS. We also compared CcpA-mediated repression to other key regulators of tst, finding that CcpA regulation is dominant compared to other characterized regulatory mechanisms. This study underlines the importance of environmental signaling for S. aureus pathogenesis in the context of mTSS. IMPORTANCE Menstrual toxic shock syndrome (mTSS) is caused by strains of Staphylococcus aureus that overproduce a toxin known as toxic shock syndrome toxin-1 (TSST-1). This work studied how glucose levels in a model vaginal environment could influence the amount of TSST-1 that is produced by S. aureus. We found that high levels of glucose repress TSST-1 production, and this is done by a regulatory protein called catabolite control protein A (CcpA). The research also demonstrated that, compared with other regulatory proteins, the CcpA regulator appears to be the most important for maintaining low levels of TSST-1 in the vaginal environment, and this information helps to understand how changes in the vaginal environmental can lead to mTSS.

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CcpA Regulates Staphylococcus aureus Biofilm Formation through Direct Repression of Staphylokinase Expression.

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CcpA Regulates <i>Staphylococcus aureus</i> Biofilm Formation through Direct Repression of Staphylokinase Expression.

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