Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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. Twiari^36,37.

1. Use virus from a single lot and store at -80 °C at a 1:1 ratio of Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS) and skim milk until use. Before viral lot storage, determine virus concentration by o-nitrophenyl-β-D-galactopyranoside (ONPG) and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) assay.
2. Determine virus viability and multiplicity of infection (MOI) by X-Gal staini....

Results

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 whether.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
1x DMEM (Dubelcco's Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate	Corning	10-017-CM
Gentamicin 50 mg/ml	Sigma	1397	50 µg/ml final concentration in the complete DMEM
Trypsin EDTA (0.05% Trypsin, 0.53 M 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
1x PBS	Dot Scientific	30042-500
RIPA Lysis	Life Technologies	89901
Staining
Methanol	Fisher Scientific	A433P-4
HSV 1&2, specific for gD	ViroStat	196
DAPI	SIGMA	D8417-5MG
Gram Crystal Violet	Troy Biologicals	212527
Supplies
Petri dish 100 x 15	Dot Scientific	229693
Petri dish 150 x 15	Kord Valmark	2902
96-Well plates	Evergreen Scientific	222-8030-01F
24-well plates	Evergreen Scientific	222-8044-01F
Culture tubes 100 x 13	Thomas Scientific	9187L61
Cover slip circles, 12 mm	Deckglaser	CB00120RA1