Large-Scale Screening and Identification of Novel Pathogenic Staphylococcus aureus Genes Using a Silkworm Infection Model

Abstract

The regulatory network of virulence factors produced by the opportunistic pathogen Staphylococcus aureus is unclear and the functions of many uncharacterized genes in its genome remain to be elucidated. In this study, we screened 380 genes whose function was unassigned, utilizing gene-disrupted transposon mutants of the community-acquired methicillin-resistant S. aureus USA300 for pathogenicity in silkworms. We identified 10 strains with reduced silkworm killing ability. Among them, 8 displayed reduced virulence in a mouse model as evidenced by reduced colony-forming units in organs of infected mice. The role of each gene in pathogenicity was further confirmed by complementation and pathogenicity tests in silkworms, where we found that the phenotype was not restored in 1 strain. Additionally, some of the mutants displayed reduced hemolysis, proteolysis, pigment production, and survival in murine RAW 264.7 monocyte-macrophage cells. These newly identified genes involved in virulence will enhance our understanding of the pathogenicity of S. aureus.

YjbH regulates virulence genes expression and oxidative stress resistance in Staphylococcus aureus

“…To identify the S. aureus virulence factors and the genes responsible for pathogenicity, we performed screening using a silkworm infection model. Over the past years, we have identified several S. aureus virulence factors that are involved in both the silkworm and mice virulence [11][12][13][14].…”

Emerging Evasion Mechanisms of Macrophage Defenses by Pathogenic Bacteria

“…In particular, heterogeneity of bacterial and macrophage populations, spatiotemporal dynamics of interactions and multiplicity of microenvironmental cues can hardly be recapitulated in standard assays using cell lines. This calls for appropriate in vivo infection models, which frequently reveals unexpected findings that were not or could not be observed in vitro (Blériot et al, 2015 ; Jones and D'Orazio, 2017 ; Gluschko et al, 2018 ; Paudel et al, 2020 ) and which are instrumental to development of innovative therapeutic strategies (Dickey et al, 2017 ; Kaufmann et al, 2018 ; Morrison, 2020 ).…”

Alteration of menaquinone isoprenoid chain length and antibiotic sensitivity by single amino acid substitution in HepT

“…The hepT gene from S. aureus was amplified using the primer sets BamF vs SalR1 and BamF vs SalR2 for Smith and RN4220 strains, respectively ( Table 2). The BamHI SalI digested PCR product was then ligated to pND50-pfbaA vector 23 digested with the same enzymes to construct pND50-pfbaA-hepT Smith and pND50-pfbaA-hepT RN4220 , respectively. The ligated plasmid was then transformed to Escherichia coli HST08 (Takara Bio) and selected on chloramphenicol plates.…”

“…To confirm the role of HepT Smith in longer chain MK biosynthesis, we cloned the hepT gene from the Smith strain and expressed it under the control of the constitutive expression promoter. 23 The plasmid thus obtained was introduced into the restriction deficient strain S.…”