Optical Tracking of Phagocytosis with an Activatable Profluorophore

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Analytical Chemistry

Optical Tracking of Phagocytosis with an Activatable Profluorophore Metabolically Incorporated into Bacterial Peptidoglycan

Yunpeng Tian,a Mingzu Yu, a Zhu Li,a Jiahuai Han,b Liu Yang,a,* and Shoufa Hana,*

a

Department of Chemical Biology, College of Chemistry and Chemical Engineering, State Key

Laboratory for Physical Chemistry of Solid Surfaces, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, and Innovation Center for Cell Signaling Network, Xiamen University; bState key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network, School of Life Sciences, Xiamen University, Xiamen, 361005, China

ACS Paragon Plus Environment

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Abstract: Phagocytosis is critical for immunity against pathogens. Prior imaging using dye-labelled synthetic beads or green fluorescent protein-expressing bacteria is limited by “always-on” signals which compromise discerning phagocytosed particles from adherent particles. Targeting cellular internalization of pathogens into acidic phagolysosomes, we herein report “turn-on” fluorescence imaging of phagocytosis with viable bacteria featuring peptidoglycans covalently modified with rhodamine-lactam responsive to acidic pH. Culturing of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) with d-lysine conjugated rhodamine-lactam and fluorescein isocyanate (FITC) leads to efficient metabolic incorporation of FITC and rhodaminelactam into bacterial peptidoglycan. E. coli and S. aureus become red-emissive upon phagocytosis into Raw 264.7 macrophages. With FITC as the reference signal, the mono- and dual- color emission allow efficient in situ distinction of ingested bacteria from extracellular bacteria. Given the ease of optical peptidoglycan labelling, the prevalence of microbial peptidoglycan, and preservation of microbial surface landscape, this approach would be of use for

investigation

on

microbial

pathogenesis

and

high-throughput

screening

of

immunomodulators of phagocytosis.

Keywords:

bacterial phagocytosis, signal activation, fluorescence imaging, peptidoglycan,

metabolic incorporation, phagolysosomal pH


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Introduction Pathogen-host interplay underlies infection and immune eradication.1 Phagocytosis whereby phagocytes capture, engulf and destroy invading microbes constitutes the fundamental defense of innate immunity. Deficient phagocytosis leads to recurrent infection in affected individuals. As such tools allowing phagocytosis imaging are of biomedical interests.2 Phagocytosis is typically detected by cellular uptake of fluorescein-labelled latex beads, dead bacteria or zymosan.2-9 Alternatively, green fluorescent protein (GFP)-expressing E. coli has been employed to measure phagocytosis.10,11 These approaches, with limited choice of bacteria, are often incapable of differentiating ingested particles from extracellular particles owing to intrinsic “always-on” fluorescence.2 To delineate bacterial infectivity, it is informative to image phagocytosis with authentic diverse bacteria over artificial micro-beads or inactivated bacteria (e.g. commercial Phagotest). In addition, techniques allows in situ real time tracking of phgocytosed bacteria in the presence of uningested bacteria are beneficial for pathogenesis investigation. Inspired by metabolic incorporation of d-lysine derivatives into bacterial cell wall,12 we herein report the covalent labelling of bacterial peptidoglycan with d-lysine conjugated rhodamine X-lactam (ROX-lactam) activatable to acidic pH to give intense fluorescence and d-lysine labelled FITC which serves as the internal signal reference (Figure 1A). ROX-lactam anchored on peptidoglycan beneath bacterial membrane readily isomerizes in acidic phagolysosomes to give intense red fluorescence, allowing facile tracking of bacterial phagocytosis (Figure 1)


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Results and Discussion Fuoresceinated particles were often employed for optical tracking of phagocytosis.2-9 With pKa∽6.7, fluoresceins display decreased fluorescence in acidic phagolysosomes. Furthermore, the “always-on” fluorescence compromises in situ differentiation of internalized bacteria within phagocytes from cell surface adhered bacteria.2 To monitor bacterial phagocytosis in a signal “turn-on” manner, we set to incorporate activatable profluorophores into the interior of live bacteria. In addition, we envisioned that trapping of optical probes within bacteria would largely maintain the integrity of microbial surface landscape, which is beneficial to reconstitute natural host cell-microbe interactions. Rhodamine-lactams, a group of nonfluorescent rhodamine derivatives featuring intramolecular spiro-lactam, exhibit acidic pH dependent signal via proton triggers opening of the lactams to give fluorescent rhodamines. We previously reported the use of rhodamine-lactams for imaging of lysosomal acidity and lysosomal pH based tumor imaging.13-22 Herein rhodamine-lactams were conjugated with bacterium-homing entities to envelope rhodamine-lactams within bacteria for reporting phagolysosomal acidity during bacterial phagocytosis. Loading of Bacteria with Triphenylphosphonium-conjugated Rhodamine-lactam Cationic lipophilic dyes effectively accumulate in mitochondria due to the negative transmembrane potential of mitochondria.23 Akin to mitochondria, viable bacteria possess transmembrane potentials critical for normal cell functions.24 We first attempted to target viable bacteria with mitochondria-trophic molecular probes. Triphenylphosphonium (TPP) is a cationic hydrophilic vector widely used to ferry various cargoes into mitochondria.25-31 Therefore TPP was conjugated with dansyl chloride and rhodmaine B-deoxylactam (RB-lactam) activatable to


4

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acidic pH22 to give RB-Dan-TPP for staining of viable bacteria driven by the transmembrane potentials. The dansyl moiety functions as an internal optical reporter whereas the rhodaminelactam is activatable to phagosomal pH upon bacterial engulfment (Figure 2A). To determine if RB-Dan-TPP could be taken up by live bacteria, Gram-negative E. coli were cultured

in

Luria-Bertani

(LB)

broth

spiked

with

RB-Dan-TPP

and

3,3’-

dioctadecyloxacarbocyanine (DIO) and then resuspended in buffer of pH 4 or 7. DIO is specific for plasma membrane. Confocal fluorescence microscopy analysis shows that DIO signal is confined to the periphery of bacteria and dansyl fluorescence is present uniformly in cytosol (Figure 2B), clearly demonstrating cytosolic accumulation of RB-Dan-TPP in E. coli. In contrast with the “always-on” dansyl signal, intense rhodamine fluorescence is observed in bacteria at pH 4.0 whereas no signal is observed in bacteria at pH 7.0 (Figure 2B). These results validate that TPP-conjugated RB-lactam could be taken up by viable bacteria and exhibits “turn-on” fluorescence within bacteria at acidic settings. We next examined uptake of RB-Dan-TPP loaded GFP- expressing E. coli by Raw 264.7 macrophages. Confocal microscopy analysis reveals low level colocalization of rhodmaine signal with GFP signal within macrophages (Figure 2C), showing leaking of physically trapped RBDan-TPP from GFP-harboring E. coli into macrophages. Phagocytosed bacteria could be inactivated by combined effects of acidic pH, hydrolases and reactive oxygen species, leading to loss of transmembrane electrochemical potentials. The leakage of RB-Dan-TPP is likely due to loss of microbial transmembrane potentials during phagocytosis and thus excludes the use of transmembrane potential-trapped dyes for phagocytosis imaging.


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Covalent Incorporation of pH Reporting Rhodmine-lactam into Bacterial Peptidoglycan To prevent dye leaking from phagocytosed bacteria, we sought to covalently attach optical reporters to peptidoglycan beneath bacterial plasma membrane. Diverse d-amino acids have been metabolically incorporated into cell wall via promiscuity of the peptidoglycan biosynthesis pathway.12,32-34 Inspired by these observations, d-lysine was conjugated with ROX-lactam to give ROX-lactam-d-Lys for covalent incorporation into bacterial cell wall. Compared with rhodmaine B (λex: 560 nm/λem: 590 nm), ROX is of red-shifted fluorescence characters (λex: 585 nm/λem: 610 nm) and thus was chosen as the reporter for phagocytosis. To access its pH responsiveness, ROX-lactam-d-Lys was spiked into buffer of pH 4.0-9.0. Fluorescence emission of the solutions was recorded over buffer pH. Figure 3 shows that ROXlactam-d-Lys is weakly fluorescent at cytosolic pH (pH 7.2) and becomes fluorescent at pH 4.06.0 which ideally matches phagolysosomal pH window. ROX-lactam-d-Lys exhibits fluorescence emission centered at 610 nm in acidic media and intensified as the buffer pH decreases, proving proton mediated fluorogenic opening of the intramolecualr lactam as described in Figure 1C. Apart from the red-shifted wavelength of ROX fluorescence which is advantageous for bioimaging, ROX-lactam is 20-30 fold brighter than rhodamine B-lactam in the range of pH 4.0-5.5 (Supporting information, Figure S2), further reinforcing the utility of ROXlactam for acidic pH imaging. E. coli and S. aureus were cultured in LB medium supplemented with ROX-lactam-d-Lys and FITC-d-Lys for 0.5 h. The bacteria were harvested by centrifugation, washed with phosphate buffered saline (PBS) and then suspended in buffer of pH 4.0-8.0. FITC signal is present in E. coli and S. aureus in media of pH 4-9, which is consistent with the “always-on” fluorescence of


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FITC. In contrast, ROX signal, absent in bacteria at alkaline pH, is observed from E. coli and S. aureus in acidic media (Figure 4), indicating effective incorporation of ROX-lactam into bacterial cell wall and acidity mediated turn-on fluorescence of ROX-lactam inside bacteria. Diverse D-amino acids have been documented for metabolic labeling of bacterial peptidoglycan.12,32-34 We further explored the efficacy of bacterial labeling with ROX-lactam-dLys over ROX-lactam-L-Lys. E. coli and S. aureus were respectively cultivated in medium spiked with ROX-lactam-d-Lys or ROX-lactam-L-Lys, and then suspended in buffer of pH 4.0 or pH 7.0. As shown in Figure S4 (Supporting information), bright fluorescence is observed in bacteria treated with ROX-lactam-d-Lys as compared to low level signal in bacteria treated with ROX-lactam-L-Lys. Consistent with reported observations,12,32-34 the results demonstrate preferential uptake of ROX-lactam-d-Lys over ROX-lactam-d-Lys by E. coli and S. aureus. Tracking Phagocytosis of Bacteria Covalently Labelled with ROX-lactam-d-Lys and FITCd-Lys With the demonstrated intra-microbial accumulation and acidic pH activatable fluorescence, E. coli and S. aureus labelled with Rox-lactam-d-Lys and FITC-d-Lys were respectively cultured with Raw 264.7 macrophages in DMEM supplemented with 10% fetal bovine serum. Raw 264.7 cell is a mouse leukaemic macrophage phagocyte cell line. Strong FITC signals were identified immediately after addition of dye-labelled bacteria whereas no ROX fluorescence could be observed (Figure 5), which is consistent with the nonfluorescent nature of ROX-lactam-d-Lys at extracellular neutral pH conditions. Intense ROX fluorescence appeared inside host cells after 12 h incubation. The bacteria-restricted FITC and ROX fluorescence within macrophages validates that covalent incorporation of dyes to peptidogly effectively prevents probe leakage


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from phagocytosed bacteria (Figure 5). ROX signal nicely colocalizes with that of Lysotracker blue DND-22 (referred to as LysoTracker) (Figure 5), which selectively stains acidic intracellular compartments. Taken together, these results prove turn-on fluorescence of rhodamine-lactam incorporated into peptidoglycan upon bacterial phagocytosis by macrophages. To verify the correlation of phagolysosomal acidity with fluorescence of ROX-lactam observed after phagocytosis, Raw 264.7 cells were first incubated with E. coli or S. aureus labelled with FITC-d-Lys and ROX-lactam-d-Lys, and then respectively treated with or without Bafilomycin A1 (BFA). BFA is a specific inhibitor of proton vacuolar ATPase pump located at phagosomal membrane, and thus effectively inhibit phagosomal acidification.35 In contrast with the bright ROX signal observed in BFA-free macrophages, no ROX fluorescence is presence in BFA-treated macrophages (Figure 6). In control experiments, ROX fluorescence was recovered from phagocytosed E. coli and S. aureus in BFA-treated macrophages resuspended at acidic buffer (pH 4.0) (Figure 6). Collectively, these results demonstrate the phagolysosomal acidity triggered turn-on fluorescence of ROX-lactam anchored in bacterial cell wall. Currently, in vitro infection of mammalian cells is widely utilized to investigate the pathogenic potentials and mechanisms by which facultative bacteria survive and grow within host cells. The “always-on” fluorescence of dye-labelled particles and GFP-expressing bacteria poses difficulties on distinguishing phagocytosed particles from these unphagocytosed, and thus data collecting and processing.10,11 As such, quenching agents are often employed to quench extra-cellular fluorescence.36 The successful “turn-on” fluorescence detection of phagocytosis of E. coli and S. aureus demonstrates the potentials of this approach for facile in situ imaging of phagocytosis without manual removal or fluorescence quenching of extracellular bacteria.


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Conclusions Phagocytosis of pathogens by host cells is critical for microbial pathogenesis and immunity. Prior imaging of phagocytosis often uses nonspecific technologies with high background signals, such as fluorescently labelled artificial particles or inactivated bacteria. To circumvent these limitations, we employ metabolic incorporation of d-lysine-conjugated rhodamine-lactam into bacterial peptidoglycan to give engineered bacteria that display “turn-on” signals in phagolysosomes by proton triggered fluorogenic isomerization of rhodamine-lactam. In addition, real time and in situ tracking of phagocytosis was achieved with dual colored E. coli and S. aureus featuring peptidoglycan-anchored FITC (served as the optical tag of bacteria) and rhodamine-lactam responsive to phagolysosomal acidity without tedious manual removal or signal quenching of unphagocytosed bacteria. The selective incorporation of profluorophore into peptidoglycan maintains the integrity of bacterial surface molecular landscape, which is beneficial for reconstitution of the natural bacteria-host cell interaction. Give the prevalence of peptidoglycans in diverse bacteria, this approach, synchronizing phagocytosis process and giveing phagocytosis specific readouts (rhodamine signals), is of broad utility to probe phagocytosis of different pathogens and possibly to screen immunomodulators on phagocytosis.


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Author information Corresponding author *To

whom

correspondence

should

be

addressed.

E-mail:

[email protected].

[email protected]; Phone: +865922181728. Xiamen University. Acknowledgement This work was supported by grants from NSF China (21305116, 21272196), 973 program 2013CB933901, Doctoral Fund of Ministry of Education of China (20130121120003); natural science foundation of Fujian Province (2014J01059, 2011J06004); PCSIRT, and an open project grant from supported by the State Key Laboratory of Natural and Biomimetic Drugs; Dr. J. Han was supported by grants from NSF China (91429301, 31420103910, 31330047, 31221065),the National Scientific and Technological Major Project (2013ZX10002-002), the Hi-Tech Research and Development Program of China (863 program; 2012AA02A201), the 111 Project (B12001), the Science and Technology Foundation of Xiamen (3502Z20130027), the National Science Foundation of China for Fostering Talents in Basic Research (J1310027) and The Open Research Fund of State Key Laboratory of Cellular Stress Biology, Xiamen University. Supporting Information Supporting information available on experimental procedures, synthesis and characterization of RB-Dan-TPP, ROX-lactam-L-Lys, and ROX-lactam-D-Lys. pH-mediated fluorescence of cells labelled with ROX-lactam-L-Lys. Supporting Information is available free of charge via the Internet at http://pubs.acs.org. The authors declare no competing financial interest.


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Figures Figure 1. Schematic of phagocytosis imaging with live bacteria featuring peptidoglycananchored fluorescein and acid activatable ROX-lactam (A). Chemical structures of FITC-d-Lys and ROX-lactam-d-Lysine (B). ROX-lactam undergoes proton-triggered opening of the spirolactam within phago-lysosomes to give red fluorescence (C). Figure 2. Cytosolic accumulation of RB-Dan-TPP in E. coli for sensing of acidic extracellular pH. A) Acidic pH mediated fluorogenic isomerization of RB-Dan-TPP; B) cytosolic accumulation of RB-Dan-TPP in bacteria and acidic pH mediate fluorescence-on of RB-DanTPP within bacteria; Bar, 5 µm. C) phagocytosis of RB-Dan-TPP loaded E. coli by Raw 264.7 macrophages. Bar, 10 µm. Figure 3. pH responsiveness of ROX-lactam-d-Lys. ROX-lactam-d-Lys was spiked into sodium phosphate buffer (100 mM, pH 4.0-9.0) to a final concentration of 1 µM. Fluorescence emission of the solutions was recorded using λex@585 nm (A) and the fluorescence intensities@610 nm were plotted over buffer pH (B). Figure 4. pH mediated fluorescence of ROX-lactam-d-Lys and FITC-d-Lys metabolically incorporated into bacteria. E. coli. (A) and S. aureus (B) were respectively cultured in LB medium supplemented with FITC-d-Lys (100 µM) and ROX-lactam-d-Lys (100 µM) for 0.5 h. The cells were collected, washed with PBS, resupended in buffers of pH 4-8 and then visualized by fluorescence microscopy. Bars, 5 µm. Figure 5. Tracking phagocytosis of ROX-lactam-d-Lys and FITC-d-Lys labelled bacteria by macrophages. E. coli (A) and S. aureus (B) prestained with ROX-lactam-d-Lys and FITC-d-Lys


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were respectively cultured with Raw 264.7 cells in DMEM. The cells were monitored for FITC signals and ROX signals over incubation time. Bars, 10 µm. Figure 6. Phagolysosomal acidity triggered turn-on fluorescence of ROX-lactam anchored on bacterial peptidoglycan. Raw 264.7 cells were respectively incubated in DMEM containing E. coli (A) or S. aureus (B) pre-labelled with ROX-lactam-d-Lys and FITC-d-Lys, and then treated with or without BFA. The cells were harvested, resuspended in sodium phosphate buffer of pH 4.0 or 7.2, and then visualized by confocal fluorescence microscopy. Bars, 10 µm.


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B

HO N

O

O

O

N HOOC O N O

N H

N H

N

O

NH3

N H

COO

N H

COO

FITC-d-Lys

ROX-lactam-d-Lys

C

S

NH3

N

N

O

N

Phagolysosome O

Extracellular

O

N O Nonfluorescent

N H

N H

H N

cell O

Highly fluorescent

Figure 1. Schematic of phagocytosis imaging with live bacteria featuring peptidoglycananchored fluorescein and acid activatable ROX-lactam (A). Chemical structures of FITC-d-Lys and ROX-lactam-d-Lysine (B). ROX-lactam undergoes proton-triggered opening of the spirolactam within phago-lysosomes to give red fluorescence (C).


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A

N

O

N N

N

N P

O

N

N H

N

Dansyl

N

Acidic pH

OS O

B

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OSO

P

N

Rhodomine

DIO

Merge

pH 4.0

pH 7.0

C

Dansyl

Rhodomine

GFP

Merge

Bright View

Figure 2. Cytosolic accumulation of RB-Dan-TPP in E. coli for sensing of acidic extracellular pH. A) Acidic pH mediated fluorogenic isomerization of RB-Dan-TPP; B) cytosolic accumulation of RB-Dan-TPP in bacteria and acidic pH mediate fluorescence-on of RB-DanTPP within bacteria; Bar, 5 µm; C) phagocytosis of RB-Dan-TPP loaded E. coli by Raw 264.7 macrophages. Bar, 10 µm.


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6000

pH 4.0

5000 4000 3000

9.0

2000 1000 0

590

600

610

620

W avelength (nm )

630

Fluorescence Indensity (a.u.)

B

A Fluorescence Indensity (a.u.)

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6000 5000 4000 3000 2000 1000 0 4

5

6

7

8

9

pH

Figure 3. pH responsiveness of ROX-lactam-d-Lys. ROX-lactam-d-Lys was spiked into sodium phosphate buffer (100 mM, pH 4.0-9.0) to a final concentration of 1 µM. Fluorescence emission of the solutions was recorded using λex@585 nm (A) and the fluorescence intensities@610 nm were plotted over buffer pH (B).


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A

ROX

FITC

Merge

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Bright view

pH 4

pH 5

pH 6

pH 7

pH 8

B

ROX

FITC

Merge

Bright view

pH 4

pH 5

pH 6

pH 7

pH 8

Figure 4. pH mediated fluorescence of ROX-lactam-d-Lys and FITC-d-Lys metabolically incorporated into bacteria. E. coli. (A) and S. aureus (B) were respectively cultured in LB medium supplemented with FITC-d-Lys (100 µM) and ROX-lactam-d-Lys (100 µM) for 0.5 h. The cells were collected, washed with PBS, resupended in sodium phosphate buffers of pH 4-8 and then visualized by fluorescence microscopy. Bars, 5 µm.


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A

ROX

FITC

LysoTracker Merge

Bright view

0h

1h

2h

B

ROX

FITC

LysoTracker Merge

Bright view

0h

1h

2h

Figure 5. Tracking phagocytosis of ROX-lactam-d-Lys and FITC-d-Lys labelled bacteria by macrophages. E. coli (A) and S. aureus (B) prestained with ROX-lactam-d-Lys and FITC-d-Lys were respectively cultured with Raw 264.7 cells in DMEM. The cells were monitored for FITC signals and ROX signals over incubation time. Bars, 10 µm.


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A

Rox

FITC

Lysotracker

Overlay

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Bright view

-BFA (pH 7.2)

+ BFA (pH 7.2)

+ BFA (pH 4.0)

B

Rox

FITC

Lysotracker

Overlay

Bright view

-BFA (pH 7.2)

+ BFA (pH 7.2)

+ BFA (pH 4.0)

Figure 6. Phagolysosomal acidity triggered turn-on fluorescence of ROX-lactam anchored on bacterial peptidoglycan. Raw 264.7 cells were respectively incubated in DMEM containing E. coli (A) or S. aureus (B) pre-labelled with ROX-lactam-d-Lys and FITC-d-Lys, and then treated with or without BFA. The cells were harvested, resuspended in sodium phosphate buffer of pH 4.0 or 7.2, and then visualized by confocal fluorescence microscopy. Bars, 10 µm.


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For TOC Only 213x131mm (300 x 300 DPI)


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