Appl Microbiol Biotechnol.2013 Oct;97(20):9029-9041

Characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agent bacterium, Francisella tularensis Schu S4 and the surrogate type B live vaccine strain (LVS).

Su, S., R. Saldanha, A. Pemberton, H. Bangar, S.A. Kawamoto, B. Aronow, T.J. Lamkin and D.J. Hassett*.

*Corresponding author requested to submit this summary:

Daniel J. Hassett, Ph.D.

Professor

Department of Molecular Genetics, Biochemistry and Microbiology

University of Cincinnati College of Medicine

231 Albert Sabin Way, Cincinnati, OH 45267-0524

Daniel.Hassett@UC.Edu

(513)-558-1154 (office)

Acknowledgements: We acknowledge support from the United States Defense Threats Reduction Agency INSIGHTS Program.

 

Abstract

Here, we constructed stable, constitutively expressed, chromosomal green (GFP) and red fluorescent (RFP) reporters in the genome of the surrogate strain, Francisella tularensis spp. holarctica LVS (herein LVS), and the select agent, F. tularensis Schu S4. A bioinformatic approach was used to identify constitutively expressed genes. Two promoter regions upstream of the FTT1794 and rpsF (FTT1062) genes were selected and fused with GFP and RFP reporter genes in pMP815, respectively. While the LVS strains with chromosomally integrated reporter fusions exhibited fluorescence, we were unable to deliver the same fusions into Schu S4. Neither a temperature-sensitive Francisella replicon nor a pBBR replicon in the modified pMP815 derivatives facilitated integration. However, a mini-Tn7 integration system was successful at integrating the reporter fusions into the Schu S4 genome. Finally, fluorescent F. tularensis LVS and a mutant lacking MglA were assessed for growth in monocyte-derived macrophages (MDMs). As expected, when compared to wild-type bacteria, replication of an mglA mutant was significantly diminished, and the overall level of fluorescence dramatically decreased with infection time. The utility of the fluorescent Schu S4 strain was also examined within infected MDMs treated with clarithromycin and enrofloxacin. Taken together, this study describes the development of an important reagent for F. tularensis research, especially since the likelihood of engineered antibiotic resistant strains will emerge with time. Such strains will be extremely useful in high-throughput screens for novel compounds that could interfere with critical virulence processes in this important bioweapons agent and during infection of alveolar macrophages.

 

ADDITIONAL TEXT

Introduction- A major hurdle in F. tularensis (Ft) pathogenesis is strains that harbor single-copy, chromosomal, constitutively expressed GFP/RFP. First, we used the surrogate LVS strain of Ft, and the human virulent strain, Schu S4. First, an optimized GFP gene, Superfolder (SF) GFP, and an optimized RFP gene, TurboRed (TR) RFP, were amplified by PCR. The PCR products SFGFP and TRRFP were fused to bioinformatically chosen promoters.  We used a bioinformatic approach to identify (i) highly and (ii) constitutively expressed genes both in vitro and in vivo. We ultimately found that the FTT1794 and FTTrpsF promoters represented the best candidates for our purposes. First, an upstream promoter fragment of locus FTT1794 was amplified with primers P1794/KpnSal5¢ and P1794/Pst3¢.  Next, an upstream promoter fragment of locus FTT1062 (PrpsF), was PCR amplified.  The PCR products P1794 and PrpsF were first cut with KpnI and PstI, and ligated with either PstI-ApaI digested SFGFP, or PstI-EcoRI digested TRRFP, respectively.  The ligation products were then cloned between KpnI and ApaI (or EcoRI) sites of pBluescriptSK+, creating plasmids pSK-P1794-SFGFP, pSK-P1794-TRRFP, pSK-PrpsF-SFGFP, and pSK-PrpsF-TRRFP. These plasmids were the source of the respective promoter-reporter fusions that were subcloned, pMP815 (Fig. 1A-1) and the mini-Tn7 vector pMP749, two single-copy integration systems.

Delivery of GFP/RFP reporter on a suicide vector   One kb fragments containing either a P1794-SFGFP or a PrpsF-SFGFP reporter were cloned into pMP815 (Fig. 1A-1), yielding plasmids pMP815-P1794-SFGFP (Fig. 1A-2) and pMP815-PrpsF-SFGFP (Fig. 1A-3). These plasmids were introduced into LVS via homologous recombination-dependent integration of the non-replicative sacB counter-selectable vector in the blaB site. Using fluorescence microscopy, E. coli DH5-a and LVS displayed fluorescence (Fig. 1B, below panels). Bacteria harboring the PrpsF reporter exhibited brighter fluorescence than those containing P1779 reporter (Fig. 1B, below panels).

While integration of the reporter fusions into the blaB site of LVS was achieved, we could not deliver the plasmid into Schu S4; we obtained only one KmR fluorescent Schu S4 transformant. Yet, integration was at the groEL site instead of the blaB site.  Thus, the KmR marker could not be recycled through a secondary homologous recombination. The lower transformation efficiency of Schu S4 combined with the inherently low rate of homologous recombination prevented the successful integration of the reporter in Schu S4.

pMP815-based plasmids with either a temperature-sensitive Francisella or a pBBR1 repliconWe attempted to increase the probability of homologous recombination by permitting the reporter donor plasmid to replicate. To allow for replication of these suicide plasmids in Francisella spp., a 3,056 bp fragment harboring a temperature-sensitive Francisella replicon repA and a FTN1451 promoter-driven Nat1 from the Francisella targetron vector pKEK1140-Nat1, was cloned into the unique blunt-end site MluI of pMP815-P1794-SFGFP (Fig. 2A), creating plasmid pMP815-P1794-SFGFP-repA (Fig. 2B). Transformation yielded thousands of Schu S4 transformants but we could not cure these plasmids by a shift in temperature. We next tested whether incorporation of the pBBR1 replicon in pMP815 can increase the overall transformation efficiency in Schu S4. Since the pBBR1 replicon supports favorable conditions for homologous recombination in F. tularensis, a 1,660-bp fragment containing the pBBR1 replicon from the vector pBBR1MCS-2  was amplified by PCR and cloned into the unique MluI sites of pMP815-P1794-SF, creating plasmid pMP815-P1794-GFP-pBBR1 (Fig. 2C). Unexpectedly, this plasmid still could not be transformed into Schu S4. Therefore, we pursued yet another experimental strategy to clone GFP/RFP reporters into Schu S4.

Use of a mini-Tn7 vector, pMP749, for expression of GFP/RFP fusions in Schu S4   The Tn7 system  consists of an unstable transposase plasmid pMP720, a mini-Tn7 transposon vector pMP749 and a gd-resolvase plasmid pMP672.  We had to replace the hyg gene of the plasmids pMP720 and pMP672 with the nat1 gene, which confers nourseothricin resistance.  To facilitate this, the plasmids pMP720 (Fig. 3-1) and pMP672 (Fig. 3-2) were first digested with NdeI and FseI to release a 637-bp 5¢ end of the hyg gene.  Next, a 570-bp nat1 gene from pTZ559 was amplified by PCR and cloned between the NdeI and FseI sites of pMP720 and pMP672 which lacked the hyg genes, yielding pMP720-Nat1 (Fig. 3-3) and pMP672-Nat1 (Fig. 3-4).  Next, the promoter P1794- or PrpsF-driven GFP and RFP reporter fusions were cloned between KpnI and EcoRI sites of pMP749 (Fig. 4-1), creating plasmids pMP749-P1794-SFGFP (Fig. 4-2), pMP749-PrpsF-SFGFP (Fig. 4-3), pMP749-P1794-TRRFP and pMP749-PrpsF-TRRFP (Fig. 4-4).

We first electroporated the transposase plasmid pMP720-Nat1 into Schu S4 and LVS strains and selecting for hygromycin-resistant transformants.  One hygR transformant was then electroporated with the plasmid pMP749-P1794-Superfolder GFP, pMP749-PrpsF-SFGFP, pMP749-P1794-TRRFP, or pMP749-PrpsF-TRRFP, respectively, and the transposon insertions were selected on media containing kanamycin.  After curing the unstable plasmid pMP720-Nat1, the kanamycin marker was deleted from the Tn7 insertion strains by the introduction of the resolvase plasmid pMP672-Nat1.  Finally, the pMP672-Nat1 plasmid was cured, and the Shu S4 and LVS with reporter insertions at the attTn7 sites were confirmed by PCR analysis and DNA sequencing. Fig. 4B demonstrated direct fluorescence microscopic analyses of bacterial cells of either E. coli DH5-a harboring plasmids pMP749-PrpsF-SFGFP or pMP749-PrpsF-TRRFP, or LVS and Schu S4 strains carrying P1794- or PrpsF-driven GFP and RFP reporters integrated at the chromosomal attTn7 site.  Similar to the results of Fig. 1B, the promoter PrpsF is much stronger than the promoter P1794 as demonstrated by the brightness of fluorescence.

Infection studies in monocyte-derived macrophages (MDMs)

MDMs infected with LVS-GFP and an isogenic mglA mutant To prove the utility of tagged LVS and Schu S4 strains, we examined replication of the LVS-GFP and its mglA mutant within MDMs by measuring fluorescent signal output (Fig. 5A).   Expectedly, the MDMs infected with the LVS strain displayed a pattern of detectable fluorescence similar to a classical growth pattern observed by F. novicida, where cells multiplied from nearly 106-1010 in 24 hr. Expectedly, the fluorescent signal also mirrored CFU over the entire growth curve (Fig. 5B). In contrast, MDMs infected with the mglA mutant exhibited only basal fluorescent signals, suggesting failed replication, and confirmed by CFU (Fig. 5B).

Antibiotics and Schu S4 survival in MDMs Phagocytosed Schu S4 PrpsF-GFP were treated with two different concentrations of clarithromycin or enrofloxacin. We next used phase contrast microscopy, Hoechst staining, GFP fluorescence and live/dead detection in Schu S4 infected MDM’s infected. Control Fig. 6A MDMs, Fig. 6B those infected with Schu S4 GFP, and those treated with 370 nM of clarithromycin (Fig. 6C), which kills both intra-/extracellular Schu S4 are shown. These cells showed no GFP expression. In contrast, in Fig. 6D, cell were treated with only 14 nM clarithromycin and <50% of the bacteria did not express GFP within infected MDMs. Fig. 6E represents infected MDMs treated with enrofloxacin (120 nM) which killed Schu S4 (no GFP expression). Finally, Fig. 6F demonstrates that 14 nM enrofloxacin treatment still allowed for ~50% killing of Schu S4.

 

Figures and Figure legends:

fig1Fig. 1. Construction of pMP815-P1794-Superfolder GFP and pMP815-PrpsF-Superfolder GFP plasmids. A. Vector pMP815 and its derivatives were used for these studies. The promoter P1794 and PrpsF-driven SFGFP were cloned into the blaB targeting suicide vector pMP815, yielding pMP815P1794-SFGFP (Fig. 1-2) and pMP815-PrpsF-SFGFP (Fig. 1-3), respectively. B. Examination for detectable fluorescence on LB or TSA-C plates by fluorescence microscopy in E. coli DH5-a and F. tularensis LVS. DH5-a harboring pBluescript SK+-reporter constructs, control plasmid (pMP815-promoterless GFP), pMP815-P1794-SFGFP, pMP815-PrpsF-SFGFP, or pMP815-PrpsF-SFRFP, and Francisella strains LVSDblaB::P1794-GFP or LVSDblaB::PrpsF-GFP were streaked out on LB or TSA-C plates, respectively, and examined for red (for RFP strains) or green (for GFP strains). To the far right below, note that only F. tularensis LVS demonstrated GFP expression because of the difficulties associated with the poor transformation/electroporation efficiency of strain Schu S4.

 

fig2Fig. 2. Construction of pMP815-based F. tularensis plasmids harboring either a temperature-sensitive (Ts) or a  pBBR1 replicon. Fragments harboring either a Ts Francisella replicon repA and FTN1451 promoter-driven Nat1 (encoding nourseothricin-resistance) from the Francisella targetron vector pKEK1140-Nat1 was cloned into the unique blunt-ended MluI site of plasmid pMP815-P1794-SFGFP (Fig. 2A), creating plasmid pMP815-P1794-SFGFP-repA (Fig. 2B). The pBBR1 replicon from the vector pBBR1MCS-2 was amplified by PCR and cloned into the unique MluI sites of pMP815-P1794-Superfolder GFP, creating plasmids pMP815-P1794-GFP-pBBR1 (Fig. 2C).

 

fig3Fig. 3. Modification of plasmids pMP720 and pMP672 by replacement of the hydromycin-resistance  gene (hyg). The hyg (hygromycin-resistance gene) of plasmids pMP720, Fig. 3-1) and pMP672, Fig. 3-2) were replaced by a 570-bp nat1 gene from pTZ559 (a gracious gift from Dr. T. Zahrt, Medical College of Wisconsin), creating pMP720-Nat1 (Fig. 3-3) and pMP672-Nat1 (Fig. 3-4).

 

fig4Fig. 4. Expression of GFP and RFP fusions in strain Schu S4 using of a mini-Tn7 vector, pMP749.  A. P1794– or PrpsF-driven GFP and RFP reporter fusions were cloned between KpnI and EcoRI sites of pMP749 (Fig. 4-1), creating plasmids pMP749-P1794-Superfolder GFP (Fig. 4-2), pMP749-PrpsF-Superfolder GFP (Fig. 4-3), pMP749-P1794-turboRed RFP (data not shown) and pMP749-PrpsF-turboRed RFP (Fig. 4-4).  B. Examination for detectable fluorescence on LB or TSA-C plates or in cell suspensions by fluorescence microscopy in E. coli DH5-a, F. tularensis LVS and Schu S4. DH5-a harboring pMP749-P1794-Superfolder GFP, pMP749-PrpsF-Superfolder GFP, or pMP749-PrpsF-turboRed RFP, Francisella strains LVS::Tn7-P1794-GFP, LVS::Tn7-PrpsF-GFP, or LVS::Tn7-PrpsF-RFP cultured on medium plates, and cell suspensions of Francisella strains Schu S4::Tn7-PrpsF-GFP and Schu S4::Tn7-PrpsF-RFP were examined for red (for RFP strains) or green (for GFP strains).

 

fig5Fig. 5. Replication and GFP fluorescence of F. tularensis LVS and an isogenic mglA mutant strain within monocyte-derived macrophages over time. Time-course studies were initiated with approximate 105 GFP-tagged wild-type strain LVS and its isogenic mglA mutant bacteria in MDMs over a period of 42 hours at various intervals (See Materials and Methods for details). Our goal was to assess GFP expression vs. colony forming units (CFU) over this time period. A. Bacterial GFP expression recorded under fluorescence microscopy after fixing the infected MDMs at the indicated time points.   B. CFU analysis in triplicate cell extracts.  Wild-type LVS multiplied within MDMs from 5.3 x 104 CFU/ml to 1.5 x 107 CFU/ml after 42 hrs.  In contrast, the mglA mutant cells exhibited very poor, if any or no growth within MDMs over the period.

 

fig6

Fig. 6. MDMs infected with GFP-tagged F. tularensis Schu S4. a) Uninfected MDMs; b) Infected MDMs; c) Infected MDMs treated with 370 nM clarithromycin; d) Infected MDMs treated with 14 nM clarithromycin; e) Infected MDMs treated with 120 nM enrofloxacin; f) Infected MDMs treated with 14 nM enrofloxacin. Seven day old MDMs in complete media (RPMI 1640, 10% heat inactivated FBS, 2 mM L-glutamine, 40 U/mL granulocyte-macrophage CSF, 100 U/mL macrophage CSF), were treated with compounds or DMSO control for 3.5 hours and then treated with a suspension with Ft Schu4 rpSF GFP in RPMI/10% human AB serum at a MOI of 50. MDMs were incubated with bacteria at 37oC, 5% CO2 for 90 minutes and then washed 3 times with PBS and treated with fresh complete media. After 30 hours cells were washed twice with PBS, treated with Live/Dead® Far Red viability stain in PBS for 30 minutes at 37oC, 5% CO2, washed once with PBS, fixed with 4% PFA at room temperature, washed once with PBS and treated with 1 mM Hoechst 33342 in PBS. Cell images were captured at 20X magnification using 4 different filter settings: nuclear (blue, 377/477 nm ex/em), phase contrast (no filter), GFP (green 485/524 nm ex/em), Live/Dead viability stain (red, 628/692 nm ex/em). Images were overlaid using Image J software.

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