Immunology and Infection
Published: July 6th, 2016
A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.
The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV's effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.
The human microbiome includes diverse organisms from multiple taxonomic kingdoms that share geographic regions in the body. Adherence to cell surfaces is an essential first step in biofilm formation, which is part of the microbiome colonization process. Included in the microbiome can be viruses that cause chronic and persistent infections. The chronic cell infection by these viruses can cause an alteration in putative receptor availability.1,2 In addition, cell entry by intracellular pathogens could also affect host membrane fluidity/hydrophobicity which in turn may alter attachment of other microbiome members, including bacteria and fungi. In order to unde....
1. HSV Strains and Handling
Note: Recombinant non-spreading HSV-1(KOS) gL86 and HSV-2 (KOS) 333gJ- with beta-galactosidase reporter activity used were provided by V. Twiari36,37.
The level of robustness of data obtainable from system described in this report is shown in Figure 2 a-f 38. Through the use of this system the modulation of staphylococcal and fungal interaction with virally infected cells and their effect on each other's adherence can be delineated. These types of studies require microscopic examination of the interaction as shown in Figures 3 and 4 38 in order to determine whe.......
Currently no information is available on complex interactions between permanent to semi-permanent members of the host microbiome that cross multiple taxonomic domains, i.e., prokaryotic, eukaryotic and viral. Therefore we developed a novel in vitro model system to study biofilm initiation by S. aureus and C. albicans on HSV-1 or HSV-2 infected HeLa 229 (HeLa) cells 38. The HeLa cell model system presents a unique advantage. This is due to their lack of surface fibronectin ex.......
|BBL Sabouraud Dextrose
|Mannitol Salt Agar
|Sheep blood agar
|1xDMEM (Dubelcco's Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate
|50µg/ml final concentration in the complete DMEM
|Trypsin EDTA (0.05% Trypsin, 0.53M EDTA)Solution 1X
|Fetal Bovine Serum
|10% final concentration in the complete DMEM
|Other medium and reagents
|Ultra-Pure X gal
|1x HBSS (Hanks' Balanced Salt Solution)
|HSV 1&2, specific for gD
|Gram Crystal Violet
|Petri dish 100X15
|Petri dish 150X15
|Culture tubes 100x13
|Cover slip circles, 12mm
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