Visible fluorescence detection of type III protein secretion from bacterial pathogens

Article abstract of DOI:10.1021/ja102257v

Type III protein secretion is essential for many Gram-negative bacterial infections of host cells and an attractive target for new antibacterial drugs. Here, we describe a bacterial protein effector-carboxypeptidase G2 (CPG2) reporter system for fluorescence and visible detection of type III protein secretion in Salmonella typhimurium. This system provides a general method for measuring protein expression and secretion as well as a high-throughput and quantitative assay for analyzing type III protein secretion inhibitors.

Full text of DOI:10.1021/ja102257v

Published on Web 05/26/2010
Visible Fluorescence Detection of Type III Protein Secretion from Bacterial
Pathogens
Jacob S. Yount,† Lun K. Tsou,† Paul D. Dossa,† Amy L. Kullas,‡ Adrianus W. M. van der Velden,‡
and Howard C. Hang*,†
Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller UniVersity, New York, New York 10065 and
Department of Molecular Genetics and Microbiology, Stony Brook UniVersity, Stony Brook, New York 11794
Received March 17, 2010; E-mail: hhang@rockefeller.edu
A number of Gram-negative bacterial pathogens utilize type III
secretion systems (T3SSs) resembling molecular needle complexes to
inject protein effectors into host cells1 for invasion and intracellular
replication.2 T3SSs are essential for bacterial virulence, but the cellular
factors that regulate T3SSs and protein secretion are not fully
understood.1 Recently described small molecules that block the
secretion of bacterial effectors through T3SSs are promising leads for
new antibacterials3 but will require further development for clinical
studies. Sensitive and high-throughput assays for type III protein
secretion should facilitate mechanistic studies of T3SSs and discovery
of small molecule inhibitors.4 Herein, we describe a carboxypeptidase
G2 (CPG2)-based reporter system for fluorescence and visible detection
of type III protein secretion from Gram-negative bacterial pathogens
such as Salmonella enterica2 (Scheme 1).
Glu-CyFur is an efficient substrate of CPG2 with a kcat ) 0.25 ( 0.03
s-1 and KM ) 1.87 ( 0.53 µM (Figure S2). CPG2 is specific for Glu-
modified substrates as an aspartic acid derivative, Asp-CyFur, was not
uncaged under identical reaction conditions (Figure 1B). The time-
dependent uncaging of Glu-CyFur with CPG2 can be readily observed
by the naked eye with or without UV-excitation (Figure 1C). These
results demonstrate Glu-CyFur is a specific and efficient substrate for
CPG2. Notably, the synthesis of Glu-modified fluorophores is concise,
high yielding, and potentially compatible with other fluorogenic dyes
(Scheme S1).
Scheme 1. A Bacterial Secreted Effector-CPG2 Fusion Protein
Reports on Salmonella Type III Secretion through Color and
Fluorescence Change of Glu-CyFur
Figure 1. (A) Fluorescence emission spectra of CyFur dyes with 563 nm
excitation. (B) Change in fluorescence at 610 nm of CyFur dyes with rCPG2.
(C) Visual change in Glu-CyFur fluorescence with rCPG2.
The CPG2:Glu-CyFur reporter system provides a unique opportunity
to monitor type III protein secretion from bacterial pathogens. To
evaluate protein secretion through the Salmonella pathogenicity island-1
(SPI-1) T3SS, CPG2 with a C-terminal HA tag was fused to the
C-terminus of an SPI-1 effector SopE2 (Figure 2A). SopE2 is secreted
by the T3SS needle complex into growth media or injected into host
cells, where its guanine nucleotide exchange factor activity activates
Rho-family GTPases to remodel the actin cytoskeleton for Salmonella
entry. For expression in S. typhimurium, SopE2-CPG2-HA was cloned
into the pWSK29 plasmid driven by the SopE2 promoter (Figure 2A).
Western blot analysis of bacterial cell lysates and concentrated growth
media demonstrated that SopE2-CPG2-HA is expressed and secreted
by S. typhimurium (Figure 2B). Importantly, secreted SopE2-CPG2-
HA was enzymatically active as measured by an increase in Glu-CyFur
fluorescence (Figure 2C). Glu-CyFur uncaging was not observed for
media from bacteria expressing a SopE2-CPG2-HA active site mutant
(E175A), even though this protein was expressed and secreted (Figure
2B,C). Confirming that this assay measures secretion through the T3SS,
S. typhimurium deficient in a structural component of the SPI-1 T3SS
needle complex (∆inVΑ)10 did not secrete SopE2-CPG2-HA and,
consequently, did not exhibit CPG2 activity in the growth media
(Figure 2B,C). Immunofluorescence analysis of infected HeLa cells
confirms that SopE2-CPG2-HA can be injected into host cells within
30 min (Figure 2D). Glu-CyFur fluorescence uncaging could also be
observed for these infected cell lysates (Figure 2E) confirming
orthogonality of CPG2 activity to eukaryotic proteomes. These results
demonstrate that SopE2-CPG2-HA is stably expressed, is readily
The orthogonal specificity of CPG2 presents an attractive enzyme
reporter system to monitor bacterial protein secretion. CPG2 is a 43
kDa metalloprotease, found in rare Pseudomonas syringae strains, that
selectively cleaves glutamate (Glu) from small molecule metabolites.5
Importantly, the glutamyl-carboxypeptidase activity of CPG2 is not
present in most species of bacteria or higher eukaryotes and has been
used clinically for methotrexate toxicity as well as for uncaging of
Glu-modified pro-drugs.5 The ability of CPG2 to hydrolyze urea
analogues of Glu provides a unique enzymatic activity to uncage
fluorogenic dyes.5 Based upon the tunable, photostable, and fluorescent
properties of 2-dicyanomethylene-3-cyano-2,5-dihydrofuran (CyFur)
fluorophores,6-8 we synthesized a Glu-modified derivative of CyFur
(Glu-CyFur) as a potential fluorogenic substrate for CPG2 (Scheme
S1). Similar to other acylated derivatives of CyFur that we have
previously sythesized,8 the absorbance and fluorescence emission
properties of Glu-CyFur are significantly altered compared to CyFur
(Figure 1A and Figure S1). The visible fluorescence emission of Glu-
CyFur at 610 nm is reduced approximately 5-fold compared to CyFur
(Figure 1A), which is consistent with previously reported quantum
yields of CyFur derivatives.6-8 Importantly, recombinant (r)CPG2
uncaged Glu-CyFur within minutes (Figure 1B), demonstrating that
† The Rockefeller University.
‡ Stony Brook University.
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10.1021/ja102257v  2010 American Chemical Society

C O M M U N I C A T I O N S
translocated by the SPI-1 T3SS, and maintains CPG2 activity after
secretion. It is important to note that while expression of SopE2-CPG2-
HA using a low copy plasmid provides a convenient readout of the
SPI-1 T3SS, chromosomal expression of SopE2-CPG2 and normaliza-
tion for CPG2 fusion will be required to accurately measure endog-
enous levels of type III protein secretion. Nonetheless, the visible
fluorescence detection of Glu-CyFur uncaging provides a very sensitive
assay for type III protein secretion in bacterial supernatants and infected
cell lysates.
Figure 3. (A and B) S. typhimurium strains were grown with varying doses
of INP0007 for 4 h. SopE2-CPG2-HA secretion and expression were
visualized by western blot and by Glu-CyFur uncaging. (C) T3SS inhibitors.
(D) Dose-dependent inhibition of SopE2-CPG2-HA secretion by T3SS
inhibitors. For all experiments equal volumes of bacterial growth media
were used and comparable bacteria growth was confirmed by measuring
optical density at 600 nm.
CPG2:Glu-CyFur reporter system in measuring differential inhibi-
tory activity of small molecules targeted at T3SSs.
Figure 2. (A) SopE2-CPG2-HA bacterial expression construct. (B) Western
blots for SopE2-CPG2-HA expression from concentrated growth media or
lysates of indicated strains. (C) Change in CyFur fluorescence over 2 h
when 10 µL of growth media from the indicated bacteria strains were added.
(D) Visualization of SopE2-CPG2-HA injection into HeLa cells. Cells were
infected for 30 min with S. typhimurium expressing SopE2-CPG2-HA and
stained with anti-HA antibodies (green) and TOPRO-3 reagent (blue). Scale
bars indicate 10 µm. (E) Glu-CyFur uncaging in S. typhimurium infected
HeLa cell lysates.
The CPG2:Glu-CyFur reporter system provides a robust and
sensitive method for monitoring protein expression and secretion.
This system is complementary and orthogonal to other enzyme
reporter systems such as luciferase and ꢀ-lactamase, making it
potentially useful for dual-imaging applications in the future.4,14
Cell-permeable versions of Glu-CyFur may also be useful for live
cell and in ViVo applications. Importantly, the CPG2:Glu-CyFur
system provides a simple and rapid means of evaluating cellular
factors that influence the secretion of protein toxins and effectors
as well as chemical inhibitors for antibiotic development.
To explore the sensitivity of the SopE2-CPG2-HA:Glu-CyFur
system, we evaluated the activity of reported bacterial T3SS inhibitors
with our assay. Salicylidene acylhydrazides (SAHs) such as INP0007
have been shown to block T3SS activity in several Gram-negative
bacterial pathogens,3 including S. typhimurium.11,12 To evaluate T3SS
inhibitors with the SopE2-CPG2-HA:Glu-CyFur system, S. typhimu-
rium was incubated with INP0007 at increasing concentrations and
SopE2-CPG2-HA secretion was measured. Western blot analysis of
SopE2-CPG2-HA expression and secretion suggests that INP0007
inhibits bacterial effector secretion at 25 µM without affecting protein
expression (Figure 3A). These results are consistent with previous
reports of INP0007 activity on proteins secreted by Salmonella using
coomassie blue staining,11,12 which we also independently confirmed
(Figure S3). Analysis of SopE2-CPG2-HA expression and secretion
measuring Glu-CyFur fluorescence suggests 5 µM of INP0007 already
significantly inhibits the secretion of SopE2 (Figure 3B) without
interfering with protein expression (Figure S4). The inhibitory activity
of INP0007 can also be visualized by the naked eye based on the change
in Glu-CyFur spectral properties after CPG2 uncaging (Figure 3A).
To investigate whether the SopE2-CPG2-HA:CyFur reporter
system could differentially measure the activity of T3SS inhibitors,
we evaluated an INP0007 analogue lacking the dibromophenol
motif (SAH-1, Figure 3C). Interestingly, SAH-1 showed no
inhibitory activity compared to INP0007, which suggests that the
dibromophenol motif of INP0007 is crucial for potent inhibition
of T3SSs (Figure 3D). INP0007 is ∼4 times more potent in blocking
SopE2-CPG2-HA secretion than another reported Gram-negative
bacteria protein secretion inhibitor, 2-imino-5-arylidene-thiazoli-
dinone (IAT),13 with IC50 values of 5.5 and 22.6 µM respectively
(Figure 3D). These experiments highlight the sensitivity of the
Acknowledgment. We thank Andreas J. Baumler (Univ. of Cal.,
Davis) for the ∆inVA strain. J.S.Y. is a postdoctoral fellow of the
Irving Institute Fellowship Program of the Cancer Research
Institute. P.D.D. thanks the Rockefeller/Sloan-Kettering/Cornell Tri-
Institutional Program in Chemical Biology. H.C.H. acknowledges
support from Starr Cancer Consortium, Lerner Family, and NBC/
NIH/NIAID (2 U54 AI057158-06).
Supporting Information Available: Experimental procedures,
supporting figures, and complete refs 7 and 10. This material is available
free of charge via the Internet at http://pubs.acs.org.
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