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 understand the interactions that can occur between these multiple pathogens that co-localize in the same geographic regions of the human host, we must be able to study the interaction of pathogens that represent the spectrum of taxonomic kingdoms present at the mucosal surface.

The Herpesviridae are a family of microbes present in 100% of humans as permanent members of the microbiome^3,4. In addition they can also be persistently shed both in the presence and absence of symptoms. Specifically, herpes simplex virus-1 and herpes simplex virus-2 (HSV-1 and HSV-2, respectively) are permanent members of the microbiome in the oronasopharynx and genital tract. In immune-competent individuals, both HSV-1 and HSV-2 cause gingivostomatitis, as well as genital herpes^5-8. At these sites, HSV causes a latent infection characterized by chronic persistent asymptomatic viral shedding⁹. Entry of HSV into cells results in alterations in surface expression of nectins, heparan sulfate, lipid rafts and herpesvirus entry mediator/tumor necrosis factor receptor (HVEM/TNFr)^10-25. These potentially represent shared receptors for some bacteria and fungi, e.g. S. aureus and C. albicans,which while opportunistic pathogens, can also reside as members of the mucosal microbiome of the oronasopharynx ^26,27. Within the oronasopharynx S. aureus and C. albicans occupy two distinct sites of colonization. In hosts with natural teeth, the oral mucosa is shared by HSV-1 and C. albicans, while the anterior nasal nares are occupied by S. aureus²⁸. However, despite in vitro findings that S. aureusadheres to mouth epithelial cells, ^29,30 S. aureus is infrequently isolated from oral specimens when normal tissue is present^29,30. Little is known concerning genital tract co-colonization niches beyond the clinical findings that S. aureus is associated with aerobic vaginitis, characterized by genital inflammation, discharge and dyspareunia, while C. albicans produces mucosal lesions similar to that observed in the oral cavity^31-35. Thus, although these members of the oral and genital microbiome cross taxonomic kingdoms little is known concerning their interaction as it impacts their ability to initiate biofilm formation through adherence to the host cell surface⁵. This protocol has been effectively applied to determine the functional consequences of co-colonization/infection.

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 staining for each experimental assay run using reporter virus entry assay, as previously described (Figure 1)¹⁴.
3. Dilute virus (Opt-MEM) to desired MOI. Fix monolayers (paraformaldehyde; 0.5 ml/well) before staining. Place virus viability controls in a separate microtiter plate in parallel with polymicrobial assay plates.

2. HeLa 299 Cell Handling

1. Grow at 37 °C, 5% CO₂ in DMEM with 4.5 g/L glucose, 10% heat-inactivated fetal bovine serum (FBS), gentamicin (50 µg/ml) and L-glutamine. Passage cells at 80% confluence with Trypsin solution (ethylenediaminetetraacetic acid, 0.53 M EDTA; 0.05% Trypsin; 5 ml/flask).

3. C. albicans Handling

Note: C. albicans obtained from a clinical laboratory source is stored at -80 °C in Remmel skim milk 2x medium.

1. Culture frozen stock onto Sabouraud Dextrose Medium (37 °C). After 24 hr subculture the C. albicans onto Fungisel medium (37 °C; 48 hr) for use.
2. Generate germ tube (GT) forms (pick representative colonies; 3 ml FBS; 3 hr; 37 °C; Abs₆₀₀, 0.3). After incubation, wash GT (HBSS; 2x; 4,000 x g). Add washed GT to warmed HBSS (37 °C; 0.32 Abs₆₀₀). GT forms should be 99% of cells observed as determined by hemocytometer count.
3. Make yeast form (YF) stock suspensions by picking representative Fungisel colonies (HBSS, 3 ml; 0.32 Abs₆₀₀). Count the number of YF forms/ml microscopically using a hemocytometer. YF forms should be 99% of cells observed as determined by hemocytometer count.
4. Make working fungal stock (250 µl of GT or YF stock in 25 ml HBSS; 37 °C; 10⁵ CFU/ml)

4. S. aureus Handling

1. Store S. aureus ATCC 25923 (-80 °C; Remmel skim milk 2x). Culture onto sheep blood agar (5%; 37 °C; 24 hr). Pick representative colonies and transfer to mannitol salts medium within 2 days for stock (37 °C; 18 hr).
2. Make S. aureus stock suspension (3 ml HBSS; 1.32 Abs_{600 ;}10⁸ CFU/ml)
   1. Make working S. aureus stock (100 µl of the stock in 25 ml HBSS; 10⁵ CFU/ml).

5. Candida and S. aureus Suspensions

1. Make mixed C. albicans and S. aureus suspension (250 µl YF or GT stock and 100 µl S. aureus stock in 25 ml HBSS).

6. Polymicrobial Biofilm Assay

1. Seed 96 well plates with 200 µl of 2 x 10⁵ HeLa cells/ml (85% confluence level). Rock plates (30 - 45 min; 37 °C) before incubation (37 °C; 5% CO₂ incubator; 18 hr). Wash monolayers (1x; Opt-MEM ) then seed with HSV (HSV-1 (KOS) gL86 or HSV-2 (KOS) 33 gJ^- in 100 µl Opt-MEM ; MOI 50 and 10). Incubate plates (3 hr; 37 °C; 5% CO₂). Use only one viral strain per day.
2. Wash infected monolayers (1x; phosphate buffered saline (PBS) with Mg⁺² and Ca⁺²; 100 µl). Replace PBS with warm HBSS leaving 25 µl in each well.
   1. Add YF, GT and/or S. aureus working suspensions (100 µl; target to cell ratio =5:1; n=16) as indicated in Table 1. Incubate plates (static; 30 min; 37 °C; 5% CO₂).
3. After incubation, aspirate one column at a time immediately refilling with 300 µl PBS with Mg⁺² and Ca⁺². Repeat this step twice then add radio-immunoprecipitation assay lysis buffer (RIPA; filter sterilized; 200 µl of a 1:50 dilution).
4. Rapidly triturate the HeLa cell lysate then place 50 µl onto mannitol salts (MS) and/or Fungisel (F) media (Figure 2). Spread the lysate using a glass rod bent at a 90° angle. Incubate the plates (18 hr at 37 °C). Manually count the number of colonies per plate. Controls consist of S. aureus and/or C. albicans adherence to HSV-uninfected HeLa cells.

7. Imaging Studies

1. Wash each round glass coverslip (12 mm; 50 ml acetone in 100 ml beaker). Dry and sterilize coverslips (Kimwipes; glass petri dishes). Place dry sterile coverslips into the wells of sterile 24 well plates with alcohol flame sterilized forceps.
2. Add HeLa cells (1 ml; 5x volume used for 96 well plates) to the wells of 24 well plates containing the washed sterile round glass coverslips. Add the virus, bacteria and fungi according to the template (Table 2) at 5x the volume used for the 96 well plates, then incubate and process as described in steps 6.2.1 to 6.4 above.
3. After the final wash, fix the cells for microscopy by flooding the slide with methanol and allowing it to evaporate. Store the plates at RT until staining.
4. For bright field microscopy (1,000x final original magnification) fill the wells containing methanol-fixed coverslips with deionized water. Immediately aspirate water. Cover each coverslip with Grams crystal violet. Wash coverslips free of non-bound stain (deionized water). Dry coverslips in situ then adhere them with hard set mounting medium to a labeled slide (Figure 3).
5. For fluorescent microscopy (100x objective; 1,000x final original magnification) wash coverslips free of methanol essentially as described in step 7.4. Dry the coverslips in the wells.
   1. After drying, remove the cover slips and place them on labeled slides. Then add a sufficient amount of 1:20 dilution of fluorescein isothiocyanate (FITC)-conjugated Herpes Simplex Virus Type 1 + 2 gD antibody to cover the coverslip (1 - 5 µl).
   2. Incubate the slides in a moisture chamber at 37 °C for 30 min. After incubation, wash the coverslips in 4 changes of PBS.
   3. After the final wash, return the coverslip to the labeled slide. Stain with 4′,6-diamidino-2-phenylindole (DAPI; moisture chamber; 37 °C for 30 min). After incubation wash the coverslips in 4 changes of PBS, then affix to the labeled slide with hard set mounting medium.
   4. Allow the mounting medium to cure for 24 hr at RT in the dark. Examine the coverslips under oil objective on either a bright field microscope or fluorescent microscope with FITC and DAPI cutoff filters. Examine pictures of at least 50 fields (100 cells per organism minimum) for co-localization (Figure 4).

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 whe...

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

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
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
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 100X15	Dot Scientific	229693
Petri dish 150X15	Kord Valmark	2902
96-Well plates	Evergreen Scientific	222-8030-01F
24-well plates	Evergreen Scientific	222-8044-01F
Culture tubes 100x13	Thomas Scientific	9187L61
Cover slip circles, 12mm	Deckglaser	CB00120RA1