Uncovering Staphylococcus aureus genes with roles in pathogenicity by silkworm infection model

Abstract

22The regulatory network of virulence factors production by Staphylococcus aureus, an 23 opportunistic pathogen, is incompletely understood, and the functions of many 24 uncharacterized genes in its genome remain to be uncovered. We screened 380 25 function unassigned genes disrupted mutants of the community-acquired methicillin-26 resistant S. aureus USA300 for pathogenicity using silkworms and identified 11 27 strains with reduced silkworm killing ability. Nine out of the 11 strains displayed 28 reduced virulence in the mouse model as evidenced by reduced colony-forming units 29 in organs of the infected mice. Three of the identified gene-disrupted mutants had 30 reduced hemolytic activity, one among the three also had reduced proteolytic activity 31 and pigment production. These results suggest that silkworm model could identify 32 the genes required for virulence in the mouse model. The newly identified genes 33 involved in virulence in this study facilitates the further understanding of the 34 pathogenicity of S. aureus. 35 36 Importance 37We performed a large scale screening of mutants of Staphylococcus aureus with 38 disruption in function unassigned genes using silkworm infection model and 39identified eleven genes required for full virulence in silkworm. Nine of the eleven 40 genes were involved in virulence in mice and were previously not known to 41 aggravate virulence of S. aureus. The results suggest that silkworm model is suitable 42 for quantitative measurement of virulence, which is shared between silkworms and 43 mammals. 44 45 48 endocarditis, and bacteremia(1). With an increasing rise in the resistance of 49 methicillin-resistant S. aureus (MRSA), the appearance of vancomycin-intermediate 50 S. aureus (VISA)(2, 3), and vancomycin-resistant S. aureus (VRSA)(4-7), S. aureus 51 has become one of the leading causes of morbidity and mortality and has been 52 categorized as a threat among antimicrobial-resistant strains by the World Health 53 Organization(8) and the Centers for Disease Control and Prevention(9). The ability of 54 S. aureus to rapidly infect host is due to the expression and release of many 55 virulence factors such as cytolysins, hemolysins, leukocidins, coagulases, adhesins, 56 proteases, nucleases, enterotoxins, lipases, exfoliative toxins, and immune-57 modulatory factors, and cell surface-associated proteins including Protein A and 58fibrinogen-, fibronectin-and collagen-binding proteins(10-15). Although some of the 59 pathways of virulence factor expression and regulation such as sarA, agr, srrAB, 60 saeRS, ArlRS,(16) in S. aureus as well as the factors acting via one or more of these 61 pathways (17)(18)(19) are studied, the understanding of the production and regulation of 62 virulence factors in S. aureus is through a multifaceted network that is still obscure 63 and many genes with unknown functions in S. aureus genome with roles in virulence 64 and pathogenicity remain to be uncovered. 65 66Since virulence is the degree of pathogenicity of a microorganism that is mostly...

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).…”

The Role of Amino Acid Substitution in HepT Toward Menaquinone Isoprenoid Chain Length Definition and Lysocin E Sensitivity in Staphylococcus aureus

“…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 (Paudel et al, 2020) 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 and RN4220 strains, and expressed under the control of the constitutive expression promoter (Paudel et al, 2020). The plasmids thus obtained were then introduced into S. aureus RN4220.…”