Based on the gene profile changes by CCG-203592, downregulation of these genes could lead to defects in biofilm formation at different stages and could also lead to diminished virulence. In conclusion, this class of novel anti-virulence compounds demonstrates inhibitory effects on gene expression of multiple S. aureus virulence factors, especially genes known to be involved in biofilm formation, resulting in significant inhibition of biofilm formation. The compounds also inhibit SK gene expression in GAS, suggesting that this class of compounds could target a gene regulatory mechanism that is conserved between GAS and S. aureus. This class of compounds could be a starting point for development of novel anti-microbial agents against multiple pathogens.
Materials and Methods Bacterial Strains and Culture Conditions
GAS strain UMAA2616 was derived from the M type 1 strain MGAS166 [64]. The laboratory bacterial strains S. aureus Newman and RN6390 were used in this study. S. aureus Newman, a human clinical strain isolated from a case of secondarily infected tubercular osteomyelitis [65], was kindly provided by Dr. Olaf Schneewind, University of Chicago. S. aureus RN6390 [66] is a strain derived from RN1 [67] which was isolated from a sepsis patient. S. aureus RN6390 were provided by NARSA, which is supported under NIAID, NIH Contract No. HHSN272200700055C. NRS234 and NRS235 are clinical isolates associated with native valve endocarditis from NARSA. The UMAA2616 strain was grown in Todd-Hewitt broth containing 0.2% yeast extract (THY) (Difco, Detroit, MI) supplemented with 100 mg/mL streptomycin [21]. Planktonic culture of S. aureus was grown in THY. The medium for growth of static biofilms was THY with 0.5% glucose. All bacterial cultures were incubated at 37uC.
Synthesis of CCG-2979 Analogs
CCG-203592 and CCG-205363 were synthesized in the Vahlteich Medicinal Chemistry Core laboratory at the University of Michigan. The procedures will be described in a separate publication (manuscript in progress).
SK Activity Assay
The SK activity assay was described previously [20]. Briefly, overnight UMAA2616 culture was diluted 1:1000 into fresh THY medium containing different concentrations of CCG-203592, CCG-205363 or DMSO and grown at 37uC to an OD600 nm = 1.0 in triplicate. Twenty ml of culture supernatant was added to 100 ml phosphate buffered saline (PBS), 10 ml human plasma (Innovative Research, Novi, MI), and10 ml S2403 (1 mg/ml) (Diapharma group Inc., West Chester, OH) and incubated at 37uC for 2 hours. SK activity was measured by OD405 nm and calculated as the percentage of SK activity compared to a DMSO control UMAA2616 culture. The experiment was performed three times to obtain the mean and standard error of means for SK activity of each treatment.
Biofilm Assay
The biofilm assay was performed using 96-well polystyrene flatbottom microtiter plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland) or on medical-grade silicone wafers (Cardiovascular Instrument Corp. Wakefield, MA). Silicone wafers were placed in wells of 12-well polystyrene flat-bottom microtiter plate (TPP Techno Plastic Products AG, Trasadingen, Switzerland).Overnight cultures of S. aureus were diluted 1:200 with fresh sterile THY medium containing 0.5% glucose. For the screening of test compounds, wells of 96-well microtiter plates were filled with 200 ml aliquots of the diluted cultures with 20 mM CCG-2979 analogs or DMSO in triplicate. For estimating IC50 (the half maximal inhibitory concentration), wells of 96-well microtiter plates were filled with 200 ml aliquots of the diluted cultures with different concentrations of CCG-203592, CCG-205363 or DMSO in triplicate. For testing the effect of CCG-203592 on biofilm formation of strains RN1, NRS234 and NRS 235, 50 mM CCG203592 was used. For measuring biofilm formation on silicone wafers (161 cm), wells of 12-well microtiter plate containing silicone wafers were filled with 1 ml aliquots of the bacterial culture with different concentrations of CCG-203592 or DMSO in triplicate. The plates were incubated overnight at 37uC [50,68]. Non-adherent bacteria were washed by PBS for three times. Biofilms attached to microtiter plates or silicone wafers were strained by crystal violet solution (Sigma-Aldrich, St Louis, MO) for 15 minutes. Excess stain was removed by washing with PBS. The crystal violet attached to biofilm samples was dissolved with ethanol. The absorbance at 595 nm was measured using SpectraMaxH spectrophotometer (Molecule Probe, Sunnyvale, CA) as the value of biofilm formation. Percentage inhibition of sample treated with the small compound was calculated against the mean of samples treated with DMSO. Experiments were repeated three times to obtain the mean and standard error of means of biofilm formation under each treatment.
Dulbecco’s modified Eagle’s medium (DMEM) (Life Technologies, Grand Island, NY) with 10% fetal bovine serum, 1% penicillinstreptomycin and 1% L-glutamine. Two hundred ml aliquots of cell suspensions (1.66104 per well) with different concentrations of CCG-203592 were plated in 96-well plates and grown for 24 hours at 37uC in 5% CO2 in quadruplicate. DMSO was used as the vehicle control. Twenty ml of CellTiter 96 AQueous One Solution reagent (Promega, Madison, WI) was added to each well and the plates were incubated for 2 hours. The absorbance at 490 nm was quantified with a SpectraMaxH spectrophotometer (Molecule Probe, Sunnyvale, CA). One hundred percent viability was set at the absorbance of the cells treated with only the vehicle (DMSO). The assay was performed three times to obtain the mean and standard error of means of cell viability.
Real Time RT-PCR
Overnight cultures of S. aureus RN6390 were diluted 1:200 with fresh sterile THY medium containing 0.5% glucose. Four ml aliquots of the diluted cultures with 50 mM CCG-203592 or DMSO were added into culture tubes (Fisher Scientific Co., Pittsburgh, PA), and then cultured overnight at 37uC in triplicate. Samples were collected by centrifugation at OD600 nm,0.5 (corresponding to ML growth phase), 0.9 (LL growth phase) and overnight (S) phase. Experiments were repeated three times. RNA in S. aureus cells was stabilized using RNAprotect Cell Reagent (Qiagen, Valencia, CA). S. aureus cells were digested by lysostaphin (Sigma, St. Louis, MO). RNA was then isolated by Trizol (Invitrogen, Carlsbad, CA) according to the manufacture’s protocol. After RNA extraction, TURBO DNA-free kit (Ambion, Austin, TX) was used to remove residual DNA contamination in the RNA samples. The purified RNA was reverse transcribed using iScriptTMcDNA Synthesis Kit (Bio-Rad, Richmond, CA). cDNA samples were quantified by real time PCR using CFX96 Real-Time PCR Detection System (Bio-Rad, Richmond, CA) and the 2(2DDCt) Method [70]. PCR primers for each tested genes were presented in Table 1. The expression levels of all selected genes were normalized using 16S rRNA as an internal standard [71].
