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AP53(S-)-RFP growth on human keratinocytes. Confluent human keratinocytes (HaCaT cells) were infected with AP53(S-)-RFP bacteria at a m.o.i. of 1. Images were taken at 2, 4, and 8 h post-infection. (a) DIC channel. (b) RFP channel. (c) Merged. Magnification, 600×.
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There are several advantages, both in vitro and in vivo, in utilizing bacteria that express a fluorescent protein. Such a protein can be transiently incorporated into the bacteria or integrated within the bacterial genome. The most widely utilized fluorescent protein is green fluorescent protein (GFP), but limitations exist on its use. Additional f...
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... prominent GAS virulence genes (srtA, fbp, hasA, slo, speB, pam, sk, covS), which for some (pAM, sk2b, speB, fbpA) were affected by insertion of the mCherry gene into the GAS AP53 strain. However, the increase in speB transcripts in AP53(S-)-RFP was insignificant compared to transcript levels in AP53(S+) (Fig. 2b). Infected cultured keratinocytes (Fig. 3) and skin from AP53(S-)-RFP-infected mice (Fig. 4) demonstrated that the AP53(S-)-RFP cells were detectable by their fluorescence properties. Keratinocyte infection by GAS AP53(S-)-RFP showed similar dynamics and host cytotoxicity to the parental GAS AP53(S-) strain (Fig. 5). AP53(S-)-RFP and control AP53(S-) were comparable in infected ...
Citations
... The panel of reporter genes available for GAS is small, with a primary focus on the firefly luciferase (ffluc) or the β-glucuronidase (gusA) for assessing promoter activities (Biswas and Scott, 2003;Park et al., 2003;Loh and Proft, 2013;Lamb et al., 2018). Only few studies report the use of fluorescent proteins (FPs), such as GFP or mCherry, in S. pyogenes (Nerlich et al., 2009;Aymanns et al., 2011;Vega et al., 2013;Liang et al., 2019). Previous studies have argued that streptococcal growth conditions, such as low oxygen supply and acidification of the growth medium by lactic acid production negatively impact GFP function (Hansen et al., 2001;Aymanns et al., 2011). ...
... Early studies on the use of GFP in S. pyogenes suggested that growth conditions, such as low oxygen and acidification of the growth medium, contradict the function of FPs (Aymanns et al., 2011). Only a few studies subsequently demonstrated the use of FPs, such as mCherry or GFP, to label GAS to monitor infection progression or determine protein localization (Nerlich et al., 2009;Vega et al., 2013;Liang et al., 2019). However, the performance of newer generations of fluorescent reporters with improved features has yet to be evaluated. ...
... Interestingly, although GFP and its derivatives have been widely used in many other Gram-negative and Gram-positive bacteria, the number of studies using fluorescence reporters in GAS is very limited Frontiers in Bioengineering and Biotechnology frontiersin.org (Nerlich et al., 2009;Aymanns et al., 2011;Liang et al., 2019). Here, we demonstrated that new generations of fluorescence proteins, such as mNeongreen and mKate2, can indeed be applied in GAS and, in combination with the new genetic toolset, could serve to understand protein localization, gene expression or protein-protein interactions. ...
Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes , we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes . The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078 . Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P 23 showing the strongest activity and the synthetic promoter P xylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes . We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS *, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.
... 111 Optimization of fluorescent staining may also include the addition of other molecules, particularly for the transportation of organic fluorophores through the cell envelope. 111 Beyond the addition of compounds to fluorescently stain bacteria, there is the potential to genetically modify bacteria to express fluorescent proteins; 112,113 in these instances, the potential problems of background fluorescence are mitigated. Bacteria modified to express fluorescent proteins are particularly well suited to applications requiring in situ analyses. ...
Rapid identification and enumeration of bacteria are critical, given the surge of antibiotic-resistance, global exchange of food products, and the use of bacteria for bioremediation, pharmaceutical, and food production. In response, a wide range of methods are being developed that can be broadly classified as nucleic acid-based, structure-based, mass spectrometry-based, and optically based. Optical methods have generated interest given the potential for rapid, non-destructive, high-throughput, and amplification-free measurements that require minimal sample preparation. This Perspective reviews optical methods, which are applied to identification, enumeration, and greater understanding of bacteria routinely and more importantly at the cutting edge of research, with the aim of identifying gaps and opportunities for development. We have focused primarily on methods that directly measure bacteria and not their effect on the sample matrix or sensing, which requires a biorecognition element (i.e., label specific to some component of the bacterium). We identify gaps in the existing techniques and avenues for innovation. Finally, we suggest the parameters that should be considered and recorded when reporting the development of existing and new methods for bacterial characterization. This Perspective is intended for physicists interested in developing new optical methods for the study of bacteria and microbiologists in need of an optical technique for bacterial applications.
Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes.
In this study, we set out to develop a comprehensive set of plasmids, promoters and reporters for S. pyogenes. We present an expansion to the current genetic toolbox that comprises new replicative and site-specific integrative plasmids. Moreover, we established a collection of constitutive promoters with a wide variety of strengths as well as a set of novel inducible regulatory elements, including a zinc-inducible promoter, an erythromycin-inducible riboswitch and an IPTG-inducible promoter that outperform previously described inducible systems in terms of tightness and inducibility. In addition, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. For this, we adapted a novel chemically defined medium called RPMI4Spy. This medium showed a highly reduced autofluorescence compared to other growth media and allowed efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which improved the generation of scarless gene deletions.
This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.
