The broad spectrum antiviral compound ST-669 restricts Chlamydial inclusion 1 development and ...

22 Pages·2014·0.9 MB·English

AAC Accepts, published online ahead of print on 28 April 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.02064-13
Copyright © 2014, American Society for Microbiology. All Rights Reserved.

1 The broad spectrum antiviral compound ST-669 restricts Chlamydial inclusion
2 development and bacterial growth and localizes to host cell lipid droplets within
3 treated cells
4 Kelsi M. Sandoz1 #, William G. Valiant1, Steven G. Eriksen1, Dennis E. Hruby2,
5 Robert D. Allen 3rd 2†, and Daniel D. Rockey1 *
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7 1Department of Biomedical Sciences, Oregon State University, Corvallis, OR, USA, d
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8 2Siga Technologies Inc., Corvallis, OR, USA. f
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11 *Author for Correspondence: Department of Biomedical Sciences, College of Veterinary c
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12 Medicine, Oregon State University, Corvallis, OR 97331-4804. Email: sm
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13 [email protected] r
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15 # Present address: Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, r
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16 Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, ,
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17 National Institutes of Health, Hamilton, MT 59840. 9

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19 †Present address: Oregon Translational Research and Development Institute, Portland, t
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22 Running title: Antibacterial activity of ST-669
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24 ABSTRACT Novel broad-spectrum antimicrobials are a critical component of a strategy
25 for combating antibiotic-resistant pathogens. In this study we explored the activity of the
26 broad-spectrum antiviral compound ST-669 for activity against different intracellular
27 bacteria, and began a characterization of its mechanism of antimicrobial action. ST-669
28 inhibits the growth of three different species of chlamydia and the intracellular bacteria
29 Coxiella burnetii in Vero and Hela cells, but not in McCoy (murine) cells. The anti- D
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30 chlamydial and anti-C. burnetii activity spectrum was consistent with those observed for n
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31 tested viruses, suggesting a common mechanism of action. Cycloheximide treatment in d
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32 the presence of ST-669 abrogated the inhibitory effect, demonstrating that eukaryotic
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33 protein synthesis is required for tested activity. Immunofluorescence microscopy m

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34 demonstrated that different chlamydiae grow atypically in the presence of ST-669, in a tp
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35 manner that suggests the compound affects inclusion formation and organization. a
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36 Microscopic analysis of cells treated with a fluorescent derivative of ST-669 a
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37 demonstrated that the compound localized to host cell lipid droplets (LDs), but not to .
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38 other organelles or the host cytosol. These results demonstrate that ST-669 affects /

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39 intracellular growth in a host-cell dependent manner and interrupts proper development A
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40 of chlamydial inclusions, possibly through a lipid droplet-dependent process. il
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41 2
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47 INTRODUCTION
48 Chlamydiae infect a broad range of animal species and cause disease in a variety of
49 tissues. In humans, sexually transmitted Chlamydia trachomatis is the most common
50 reportable infection in the United States, and the pathogen is a leading cause of
51 preventable blindness worldwide (1). These obligately intracellular bacteria attach and
52 enter host cells as non-replicating elementary bodies (EB) that differentiate into D
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53 metabolically active reticulate bodies (RB) once inside the cell (2). All chlamydial growth n
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54 and development occurs within an intracellular parasitophorous vacuole termed the d
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55 inclusion. Chlamydiae recruit and modify Golgi-derived vesicles to initiate inclusion
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56 growth and transport host derived phospholipids, cholesterol and fatty acids into the m

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57 inclusion and bacterial membranes (3-9). Lipids are trafficked from the site of synthesis tp
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58 to various destinations within the cell, via either a vesicle-dependent or -independent a
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59 pathway. However, recently published data suggest that chlamydiae utilize an additional a
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60 pathway for recruitment of lipids to facilitate membrane development during chlamydial .
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61 growth. Chlamydia spp. secrete proteins into the cytoplasm that localize to the surface of /

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62 lipid droplets (LDs) and translocate the LDs across the inclusion membrane. At least A
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63 40% of chlamydial phospholipid content is host derived and the blocking of LD formation il
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64 or phospholipid uptake with chemical inhibitors has a dramatic effect on chlamydial 2
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65 growth (4, 10, 11). Collectively, this provides evidence for the use of an alternate, non- 9

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66 vesicle mediated source of host derived phospholipids by chlamydiae, and demonstrates
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67 the importance of lipid trafficking and lipid acquisition for chlamydial growth and e
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68 development.
69 LDs are lipid storage organelles encased in a phospholipid monolayer, making them
70 unique from other intracellular compartments. They are prominent structures in
71 adipocytes, mammary, and liver cells, but can be abundant in other cell types as well
72 (12). LDs function in energy metabolism, steroid biosynthesis, coordination of the
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73 immune response, and serve as a reservoir of membrane lipid precursors (13-15). They
74 are targeted by both bacteria and viruses as a source of cholesterol, phospholipid, or
75 fatty acids (16). Hepatitis C virus and Dengue virus are among several viruses that have
76 been shown to utilize host LDs during their life cycle (17, 18). A diverse and increasing
77 number of intracellular organisms rely upon LD organelles during intracellular growth
78 (19-21). D
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79 ST-669 is one of several acylthiourea-based broad-spectrum antiviral molecules that n
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80 were identified and characterized by researchers at Siga Technologies in a high- d
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81 throughput screen of a chemically defined compound library (22). The compound has a
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82 high selective index and inhibits a wide variety of viruses including Dengue, HIV, and m

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83 Vaccinia viruses at submillimolar concentrations. The broad spectrum activity of this tp
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84 compound and specificity to primate cell lines suggested it may affect a host cell process a
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85 that intersects in some way with the growth and development of a wide range of a
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86 intracellular pathogens. For this reason, it was hypothesized that ST-669 may also .
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87 delay the growth and development of intracellular bacteria such as Chlamydia spp. and /

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88 Coxiella burnetii. To explore this possibility, we evaluated ST-669 against a variety of A
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89 intracellular and extracellular bacteria. The results demonstrate that ST-669 delays il
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90 growth and development by Chlamydiae and C. burnetii, and that a fluorescent 2
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91 derivative of the compound (ST-669F) localizes specifically to intracellular LDs. 9

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92
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93 MATERIALS AND METHODS e
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94 Reagents and antibodies
95 C6-NBD-ceramide (cat# N-1154), MitoTracker (cat# M-7512), and Texas Red dextran
96 (cat# D-1864) were purchased from Invitrogen. Native ST-669 and its fluorescent
97 derivative were purchased through SIGA technologies (Corvallis, OR).
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98 Cells infected with Chlamydia spp. or Coxiella burnetii were labeled with monoclonal
99 or polyclonal primary antibodies specific to chlamydial LPS, C. trachomatis L2 IncA, C.
100 caviae GPIC IncA, or a polyclonal antisera against heat-fixed C. burnetii. Appropriate
101 species and isotype-specific secondary antibodies were purchased from Invitrogen.
102 These secondary antibodies were tagged with either Alexafluor 488 (green) or Alexafluor
103 594 (red). D
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104 Host cells, chlamydial strains, and cultures n
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105 Three different cell lines were used for analysis in inhibition assays; McCoy (murine d
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106 epithelial), HeLa (human endocervical), and Vero (African green monkey kidney). All
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107 cells were propagated in Minimum Essential Medium supplemented with 10% fetal m

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108 bovine serum (MEM-10). Chlamydial strains used for assays were C. caviae GPIC, C. tp
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109 trachomatis L2 434/Bu, and C. muridarum Weiss, and all were cultured in MEM-10 a
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110 containing 10 μg/ml gentamicin. All experiments with Coxiella burnetii used the avirulent .a
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111 Nine Mile phase II strain, and were conducted with standard BSL II practices (23). .o
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112 Infections /
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113 Mammalian cells were plated in 24-well trays and incubated overnight to a confluence of A
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114 100%. Bacterial strains were infected onto cells at a multiplicity of infection of 1. Inocula il
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115 were suspended in MEM-10 containing 10 (cid:541)M ST-669 or DMSO and plated in 1 mL 2
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116 volumes. Plates were centrifuged at 2,000 rpm at 37°C for 1 h and then transferred to a 9

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117 37°C incubator.
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118 Quantification of bacterial genome copies from infected cell culture s
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119 McCoy (murine) Vero (primate) or HeLa (human) cells were plated in 24-well trays and
120 incubated overnight to a confluence of 100%. Bacterial strains were suspended in
121 MEM-10 and inoculated onto cells at a MOI of 0.5-1. Plates were centrifuged at 2,000
122 rpm at 37 °C for 1 h and then transferred to a 37 °C incubator. Infected cells were briefly
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123 sonicated to lyse and release bacteria. The lysates were collected in microfuge tubes
124 and stored at -20 °C prior to analysis. DNA was extracted using the Qiagen DNeasy
125 Blood and Tissue kit, using instructions provided by the manufacturer. The single
126 exception to the described technique was the addition of 5 mM DTT to the initial lysis
127 buffer. All Taqman reagents for quantitative real time PCR were purchased from Applied
128 Biosystems, Taqman universal PCR Master mix was used with an input of 5 μl of D
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129 template DNA and primers and probes with 5’ 6FAM and a 3’ MGBNFQ labels. n
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130 Primers/probes were specific to either C. caviae GPIC ompA (probe 6FAM- d
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131 CATCACACCAAGTAGAGC-MGBNFQ), C. trachomatis ompA (F- f
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132 CATGGTATCTCCGAGCTGACC, R-ACTGTCTTTGATGTTACCACTCTGAAC, probe

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133 6FAM-CTAGCTTTCACATCGCC-MGBNFQ), or C. burnetii dotA (24). Ten-fold serial tp
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134 dilutions of quantitated plasmid standards containing each target gene were included in a
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135 the analyses at concentrations ranging from 1 x 109 to 1 x 103 genome copies per assay. a
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136 Genome copy number and standard error were automatically calculated using an ABI .o
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137 StepOne Real Time PCR machine with standard curve settings and extrapolated to /
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138 reflect total copy number of bacteria per ml. A
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139 Immunofluorescence and cell structure labeling il
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140 C6-NBD-ceramide and ST-669F – Vero cells were seeded onto coverslips at 30% 2
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141 confluency in MEM-10 and incubated overnight. Medium was aspirated and replaced 9

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142 with 500 μl of 10 μM C6-NBD-ceramide in PBS. Plates were incubated for 1 h at 37°C, g
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143 the C6-NBD-ceramide label was aspirated and cells were overlayed with MEM-10 and s
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144 incubated at 37°C for an additional 2 h. Fresh medium containing 10 μM ST-669F was
145 added onto cells and incubated 1 h. Medium was removed and replaced with PBS
146 before mounting coverslips onto slides and examining cells on the fluorescent
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147 microscope. The C6-NBD-ceramide was visualized on the fluorescein channel and ST-
148 669F was visualized on the DAPI channel.
149 Mitotracker and ST-669F – Vero cells were seeded onto coverslips in 24-well trays at a
150 confluency of 30% and incubated overnight. Medium was removed and then replaced
151 with serum-free medium containing a mixture of 125 nM Mitotracker (Invitrogen) and 10
152 μM ST-669F, and incubated for 1 h. Medium was removed, cells were washed with PBS D
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153 and coverslips were mounted to visualize on the microscope using the rhodamine n
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154 (Mitotracker) or DAPI (ST-669F) channel. d
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155 Bodipy 493/503 and ST-669F – Vero, McCoy, or Hela cells were seeded at a confluency f
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156 of 30% onto coverslips in MEM-10 containing 100 μM oleic acid, and then incubated

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157 overnight. Medium was removed and replaced with MEM-10 containing 1 μM Bodipy tp
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158 493/503 and 10 μM ST-669F. Cells were incubated for 1 h at 37°C, washed with 1x a
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159 PBS and then mounted onto slides for microscopy. Images were visualized using the s
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160 fluorescein (Bodipy 493/503) or DAPI (ST-669F) channel. o
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161 Immunofluorescence – Cells were plated onto 12 mm coverslips in 24-well trays, o
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162 infected as above, fixed with 100% methanol for 10 min and washed twice with PBS. A
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163 Fixed cells were labeled with specific antibodies, as indicated, followed by appropriate il
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164 secondary antibodies conjugated with rhodamine or fluorescein fluorescent labels. 0
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165 RESULTS AND DISCUSSION
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166 ST-669 delays the growth of tested obligate intracellular bacteria. ST-669 is a g
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167 novel antiviral compound that has a spectrum of activity across a wide range of different s
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168 virus families (22). The current study was undertaken to examine whether ST-669 also
169 has activity against intracellular bacteria, with a primary focus on Chlamydiae and
170 Coxiella burnetii. Treatment of infected primate-derived cells with 10 μM ST-669 slowed
171 the growth of C. trachomatis, C. caviae, C. muridarum and Coxiella burnetii. Markedly
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172 smaller inclusions (C. caviae, Figures 1A,C; C. trachomatis 2A) or vacuoles (C. burnetti;
173 data not shown) are seen in ST-669 treated wells, and there is a greater reduction in
174 genome copies in ST-669 treated wells at later points during the infection (Figure 3).
175 Reduction of EB production in ST-669-treated C. trachomatis and C. caviae-infected
176 Vero cells (not shown) were parallel with the reduction in genome copies, suggesting
177 ST-669 is not bactericidal and that RB to EB transition occurs in the presence of ST-669. D
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178 These results extend the spectrum of activity of ST-669 to include these intracellular n
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179 bacteria, and increase the possible therapeutic spectrum of ST-669 or derived d
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180 compounds.
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181 m

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182 Antibacterial activity of ST-669 activity is host-species dependent. To determine if tp
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183 the antibacterial activity of ST-669 is specific to certain host cell types, we cultured C. a
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184 caviae, C. muridarum, C. trachomatis, and C. burnetii in cells of murine origin (McCoy), a
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185 primate (Vero), or human origin (HeLa) (Figure 3). ST-669 was active in HeLa and Vero .
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186 cell lines (HeLa data not shown). No inhibition of bacterial growth was observed in the /

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187 McCoy cell lines (Figure 3). These data demonstrate that the inhibitory activity of ST- A
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188 669 is host-cell-specific, which is consistent with the antiviral properties of the il
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189 compound. 2
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190 9

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191 Chlamydia caviae inclusion morphology within treated cells. Typical development
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192 of C. caviae inclusions within any tested cell line results in multi-lobed inclusions that can e
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193 be clearly visualized by staining with antibodies specific to the inclusion membrane
194 protein IncA (Figure 1 B,D; reference (25). This is in contrast to inclusions formed by C.
195 trachomatis, which are generally single inclusions containing many developmental
196 forms. Treatment of C. caviae-infected Vero cells with ST-669 led to the formation of
197 univacuolar inclusions, with chlamydiae localized to the cell in a single, round vesicle
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198 (Fig. 1A, C). This change remained consistent throughout the C. caviae developmental
199 cycle. C. caviae-infected cells treated with ST-669 also contain IncA-laden empty
200 vesicles (secondary inclusions; Fig. 1A) that are connected to the primary inclusion
201 within the cell. Secondary inclusions linked to the primary inclusion by IncA-laden fiber-
202 like structures are also observed in most strains of C. trachomatis grown in cell culture
203 (26). Therefore, treatment of C. caviae-infected cells with ST-669 leads to alteration in D
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204 inclusion structure that parallels the reduction in chlamydial genome copies, and leads to n
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205 inclusions that are similar to those formed by C. trachomatis. d
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206 Cycloheximide is an inhibitor of host protein synthesis that has no effect on bacterial
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207 translation. This distinction allowed us to examine whether host protein synthesis was m

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208 required for the activity of ST-669. An intermediate inclusion phenotype (Figure 5 C) tp
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209 and an intermediate number of chlamydial genome copies (Figure 6) were observed in a
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210 C. caviae GPIC infected cells treated with both ST-669 and cycloheximide, and this a
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211 effect was specific to HeLa and Vero cells (Figure 6 and data not shown). This suggests .
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212 that host protein synthesis is at least partially required for ST-669-mediated delay of /

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213 bacterial growth and supports the hypothesis that ST-669 mechanism of action is A
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214 directed at a host process important for chlamydial replication or development within il
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215 HeLa and Vero cell lines. 2
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216 9

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217 A fluorescent analog of ST-669 localizes to lipid droplets. A fluorescent fluorophore
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218 was conjugated to ST-669 and used to track localization of the molecule in vitro. This e
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219 derivative compound (ST-669F) has demonstrable antiviral activity against vaccinia virus
220 and C. caviae that is similar to that of the parental compound (data not shown).
221 Fluorescent antibody images of ST-669F-treated cells show a distinct localization of the
222 compound to round vesicular structures within the cytoplasm of the cell (Figures 7,8).
223 Double label fluorescence microscopy with probes for individual organelles in
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224 combination with ST-669F was used to determine subcellular compound localization.
225 There was no colocalization of labels when cells were doubly labeled with ST-669F and
226 markers that localize to lysosomes (Fig. 7A), mitochondria (Fig. 7B) or the Golgi
227 apparatus (Fig. 7C). However, Bodipy 493/503, which targets phospholipids and LDs,
228 and ST-669F colocalized very specifically within treated cells (Fig. 7D). This result
229 supports the conclusion that ST-669F localizes to LDs within treated cells. ST-669F D
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230 colocalizes with LD structures in McCoy, HeLa, and Vero cells (Figure 8), even though n
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231 antimicrobial activity is not evident in McCoy (murine) cells. Treatment of mammalian d
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232 cells with oleic acid induces LD formation, leading to expanded numbers of intracellular
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233 LDs (25). Cells treated with oleic acid show an abundance of ST-669F-labeled m

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234 structures that co-localize with Bodipy 493/503 labeled LDs (Figure 8), in both McCoy tp
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235 and Vero cells. No obvious differences are noted in LD size or morphology in ST-669 a
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236 treated cells, which is different from other chemical compounds that inhibit chlamydial a
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237 growth by blocking LD formation (10). .
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238 The colocalization of fluorescent ST-669 with LDs supports a possible model in which /

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239 ST-669 disrupts growth of both bacterial and viral organisms through a LD-mediated A
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240 mechanism, by targeting a host LD-associated pathway that is specific to HeLa and Vero il
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241 cells. A connection between lipid metabolism and ST-669 activity is also supported by 2
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242 the marked alteration in C. caviae inclusion structure in treated primate cells. This 9

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243 difference is evident very early in development and remains evident throughout the
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244 development of the pathogen. e
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245 It is also possible that ST-669 affects cells through processes independent of LD
246 biology, and that the localization of the fluorescent ST-669 compound to LDs is not
247 directly associated with its anti-infective activity. The Work by Roshick and colleagues
248 has demonstrated that innate antichlamydial immune effectors function differently in
249 murine and primate cells types (27), and some aspect of these primate-specific