Source: Plotkin, B. J. et al., Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi. J. Vis. Exp. (2016)
This video illustrates the initiation of polymicrobial biofilm formation on virus-infected cells by bacteria and fungi. Herpes simplex virus infection impedes bacterial attachment to the cells, preventing bacterial-fungal co-localization. In contrast, uninfected cells display bacterial-fungal co-localization and initiation of biofilm formation.
1. HSV Strains and Handling
NOTE: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ– with beta-galactosidase reporter activity is used for the assay.
2. HeLa 299 Cell Handling
3. C. albicans Handling
NOTE: C. albicans obtained from a clinical laboratory source is stored at -80 °C in Remmel skim milk 2x medium.
4. S. aureus Handling
5. Candida and S. aureus Suspensions
6. Polymicrobial Biofilm Assay
Table 1. General Template Determination of Microbial Adherence in Polymicrobial Interactions. GT= Candida albicans germ tube phenotype; YF= C. albicans yeast form phenotype; MSSA= methicillin-sensitive Staphylococcus aureus; MS= mannitol salts medium; F= Fungisel medium.
Figure 1. X-gal Staining Pictures of Dosage Dependent HSV-1 Infection in HeLa Cells. HeLa cells infected with HSV-1 at various MOI and X-Gal stained. (A) HeLa cells in well of 96 well plate with X-Gal stained mock-infected HeLa cell control; 20x initial magnification; (B) HeLa cells in well of 96 well plate with X-Gal stained HeLa cell infected with HSV-1 at an multiplicity of infection (MOI) of 10; 20x initial magnification; (C) HeLa cells in well of 96 well plate with X-Gal stained HeLa cell infected with HSV-1 at an multiplicity of infection (MOI) of 50; 20x initial magnification; scale bar applies to all.
Figure 2. Effect of HSV-1 (panels A, C, E) and HSV-2 (panels B, D, F) at Multiplicities of Infection (MOI) of 50 and 10 on Adherence of S. aureus and/or C. albicans to HeLa Cells. (A) S. aureus (Sa) binding to HSV-1 infected cells in the presence of C. albicans germ tubes (GT) or yeast forms (YF); (B) S. aureus (Sa) binding to HSV-2 infected cells in the presence of C. albicans germ tubes (GT) or yeast forms (YF); (C) C. albicans germ tubes (GT) binding to HSV-1 infected cells in the presence of S. aureus (Sa); (D) C. albicans germ tubes (GT) binding to HSV-2 infected cells in the presence of S. aureus (Sa); (E) C. albicans yeast forms (YF) binding to HSV-1 infected cells in the presence of S. aureus (Sa); (F) C. albicans yeast forms (YF) binding to HSV-2 infected cells in the presence of S. aureus (Sa). All data points are Mean +/- SEM, n= 16 normalized to virus-free control. * = significantly different (p< 0.05) from uninfected HeLa cell control. # = significantly different (p< 0.05) from paired point indicated by bracket; scale bar applies to all.
The authors have nothing to disclose.
C.albicans | |||
BBL Sabouraud Dextrose | BD | 211584 | |
Fungisel Agar | Dot Scientific | 7205A | |
S.aureus | |||
Mannitol Salt Agar | Troy Biologicals | 7143B | |
Sheep blood agar | Troy Biologicals | 221239 | |
Hela cells | |||
1xDMEM (Dubelcco's Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate | Corning | 10-017-CM | |
Gentamicin 50mg/ml | Sigma | 1397 | 50µg/ml final concentration in the complete DMEM |
Trypsin EDTA (0.05% Trypsin, 0.53M EDTA)Solution 1X | Corning | 25-052-CI | |
Fetal Bovine Serum | Atlanta Biologicals | S11150 | 10% final concentration in the complete DMEM |
Other medium and reagents | |||
ONPG | Thermo Scientific | 34055 | |
Ultra-Pure X gal | Invitrogen | 15520-018 | |
1x HBSS (Hanks' Balanced Salt Solution) | Corning | 20-021-CV | |
1XPBS | Dot Scientific | 30042-500 | |
RIPA Lysis | Life Technologies | 89901 | |
Supplies | |||
96-Well plates | Evergreen Scientific | 222-8030-01F | |
Culture tubes 100×13 | Thomas Scientific | 9187L61 |