Experimentally evolved Staphylococcus aureus shows increased survival in the presence of Pseudomonas aeruginosa by acquiring mutations in the amino acid transporter, GltT.

When cultured together under standard laboratory conditions Pseudomonas aeruginosa has been shown to be an effective inhibitor of Staphylococcus aureus. However, P. aeruginosa and S. aureus are commonly observed in coinfections of individuals with cystic fibrosis (CF) and in chronic wounds. Previous work from our group revealed that S. aureus isolates from CF infections are able to persist in the presence of P. aeruginosa strain PAO1 with a range of tolerances with some isolates being eliminated entirely and others maintaining large populations. In this study, we designed a serial transfer, evolution experiment to identify mutations that allow S. aureus to survive in the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa PAO1. After eight coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompeted wild-type S. aureus when glutamate was absent from chemically defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two are cultured together.

Evaluating the Performance of Pathogen-Targeted Positron Emission Tomography Radiotracers in a Rat Model of Vertebral Discitis-Osteomyelitis.

BACKGROUND: Vertebral discitis-osteomyelitis (VDO) is a devastating infection of the spine that is challenging to distinguish from noninfectious mimics using computed tomography and magnetic resonance imaging. We and others have developed novel metabolism-targeted positron emission tomography (PET) radiotracers for detecting living Staphylococcus aureus and other bacteria in vivo, but their head-to-head performance in a well-validated VDO animal model has not been reported. METHODS: We compared the performance of several PET radiotracers in a rat model of VDO. [11C]PABA and [18F]FDS were assessed for their ability to distinguish S aureus, the most common non-tuberculous pathogen VDO, from Escherichia coli. RESULTS: In the rat S aureus VDO model, [11C]PABA could detect as few as 103 bacteria and exhibited the highest signal-to-background ratio, with a 20-fold increased signal in VDO compared to uninfected tissues. In a proof-of-concept experiment, detection of bacterial infection and discrimination between S aureus and E coli was possible using a combination of [11C]PABA and [18F]FDS. CONCLUSIONS: Our work reveals that several bacteria-targeted PET radiotracers had sufficient signal to background in a rat model of S aureus VDO to be potentially clinically useful. [11C]PABA was the most promising tracer investigated and warrants further investigation in human VDO.

Experimentally Evolved Staphylococcus aureus Survives in the Presence of Pseudomonas aeruginosa by Acquiring Mutations in the Amino Acid Transporter, GltT.

Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacterial pathogens isolated from cystic fibrosis (CF) related lung infections. When both of these opportunistic pathogens are found in a coinfection, CF patients tend to have higher rates of pulmonary exacerbations and experience a more rapid decrease in lung function. When cultured together under standard laboratory conditions, it is often observed that P. aeruginosa effectively inhibits S. aureus growth. Previous work from our group revealed that S. aureus from CF infections have isolate-specific survival capabilities when cocultured with P. aeruginosa. In this study, we designed a serial transfer evolution experiment to identify mutations that allow S. aureus to adapt to the presence of P. aeruginosa. Using S. aureus USA300 JE2 as our ancestral strain, populations of S. aureus were repeatedly cocultured with fresh P. aeruginosa strain, PAO1. After 8 coculture periods, S. aureus populations that survived better in the presence of PAO1 were observed. We found two independent mutations in the highly conserved S. aureus aspartate transporter, gltT, that were unique to evolved P. aeruginosa-tolerant isolates. Subsequent phenotypic testing demonstrated that gltT mutants have reduced uptake of glutamate and outcompete wild-type S. aureus when glutamate is absent from chemically-defined media. These findings together demonstrate that the presence of P. aeruginosa exerts selective pressure on S. aureus to alter its uptake and metabolism of key amino acids when the two bacteria are cultured together.

Antigen-Dependent Inducible T-Cell Reporter System for PET Imaging of Breast Cancer and Glioblastoma.

For the past several decades, chimeric antigen receptor T-cell therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Methods: Using synthetic biology, we engineered T cells with a chimeric receptor synthetic intramembrane proteolysis receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette on recognition of specific tumor markers. We then applied a SNIPR-based PET reporter system to 2 cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors, respectively. Results: Antigen-specific reporter induction of the SNIPR PET T cells was confirmed in vitro using green fluorescent protein fluorescence, luciferase luminescence, and the HSV-TK PET reporter with 9-(4-18F-fluoro-3-[hydroxymethyl]butyl)guanine ([18F]FHBG). T cells associated with their target antigens were successfully imaged using PET in dual-xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII- animal models, with more than 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. Conclusion: The main innovation found in this work was PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.

Pseudomonas aeruginosa Antivirulence Strategies: Targeting the Type III Secretion System.

The Pseudomonas aeruginosa type III secretion system (T3SS) is a complex molecular machine that delivers toxic proteins from the bacterial cytoplasm directly into host cells. This apparatus spans the inner and outer membrane and employs a needle-like structure that penetrates through the eucaryotic cell membrane into the host cell cytosol. The expression of the P. aeruginosa T3SS is highly regulated by environmental signals including low calcium and host cell contact. P. aeruginosa strains with mutations in T3SS genes are less pathogenic, suggesting that the T3SS is a virulence mechanism. Given that P. aeruginosa is naturally antibiotic resistant and multidrug resistant isolates are rapidly emerging, new antibiotics to target P. aeruginosa are needed. Furthermore, even if new antibiotics were to be developed, the timeline between when an antibiotic is released and resistance development is relatively short. Therefore, the concept of targeting virulence factors has garnered attention. So-called "antivirulence" approaches do not kill the microbe but instead focus on rendering it harmless and therefore unable to cause damage. Since these therapies target a particular system or pathway, the normal microbiome is unlikely to be affected and there is less concern about the spread to other microbes. Finally, and most importantly, since any antivirulence drug does not kill the microbe, there should be less selective pressure to develop resistance to these inhibitors. The P. aeruginosa T3SS has been well studied due to its importance for pathogenesis in numerous human and animal infections. Thus, many P. aeruginosa T3SS inhibitors have been described as potential antivirulence therapeutics, some of which have progressed to clinical trials.

Telemedicine Intensive Care Unit (Tele-ICU) Implementation During COVID-19: A Scoping Review.

Telemedicine intensive care unit (Tele-ICU) programs entail command centers staffed with intensivists and critical care nurses who electronically aid with and deliver real-time information to frontline clinicians. The benefits of Tele-ICU are numerous, but the barriers to it often prove insurmountable, accounting for slow adoption in rural and underserved areas where it is needed the most. Remote monitoring can quickly detect patient deterioration, while consultation provided by a remote intensivist expands the capabilities of smaller facilities. The emergence of the coronavirus disease 2019 (COVID-19) pandemic has brought about a sense of urgency, paving the way for the successful adaptation of tele-intensive care concepts. The goal of this scoping review is to map out the available published data regarding healthcare professionals' experiences with implementing Tele-ICU modalities during the COVID-19 pandemic. A primary literature search was performed on PubMed/MEDLINE and the Cumulative Index to Nursing and Allied Health Literature (CINAHL) databases from October 2020 to October 2021. Of the 1,083 records screened, 19 were identified as meeting our inclusion criteria and selected for the final scoping review. Five major areas of Tele-ICU use were identified: teleconsultation, telerounding, telemonitoring, family visitation via teleconference, and changing of hospital infrastructure. A heterogeneous mix of improvised Tele-ICU platforms emerged with a common theme of interdisciplinary and family collaboration in the care of critically ill patients. Existing Tele-ICU systems were expanded, and novel programs were launched. A groundbreaking national network in the U.S. (NETCCN) will standardize the deployment of Tele-ICU and expand its reach. Future research should focus on determining accurate costs and the most reliable forms of remote communication, physician compact agreement licensure, the practical composition of Tele-ICU teams, and standardized access to the electronic health record.

11C-Para-aminobenzoic acid PET imaging of S. aureus and MRSA infection in preclinical models and humans.

Tools for noninvasive detection of bacterial pathogens are needed but are not currently available for clinical use. We have previously shown that para-aminobenzoic acid (PABA) rapidly accumulates in a wide range of pathogenic bacteria, motivating the development of related PET radiotracers. In this study, 11C-PABA PET imaging was used to accurately detect and monitor infections due to pyogenic bacteria in multiple clinically relevant animal models. 11C-PABA PET imaging selectively detected infections in muscle, intervertebral discs, and methicillin-resistant Staphylococcus aureus-infected orthopedic implants. In what we believe to be first-in-human studies in healthy participants, 11C-PABA was safe, well-tolerated, and had a favorable biodistribution, with low background activity in the lungs, muscles, and brain. 11C-PABA has the potential for clinical translation to detect and localize a broad range of bacteria.

Direct signaling of TL1A-DR3 on fibroblasts induces intestinal fibrosis in vivo.

Tumor necrosis factor-like cytokine 1A (TL1A, TNFSF15) is implicated in inflammatory bowel disease, modulating the location and severity of inflammation and fibrosis. TL1A expression is increased in inflamed mucosa and associated with fibrostenosing Crohn's disease. Tl1a-overexpression in mice causes spontaneous ileitis, and exacerbates induced proximal colitis and fibrosis. Intestinal fibroblasts express Death-receptor 3 (DR3; the only know receptor for TL1A) and stimulation with TL1A induces activation in vitro. However, the contribution of direct TL1A-DR3 activation on fibroblasts to fibrosis in vivo remains unknown. TL1A overexpressing naïve T cells were transferred into Rag-/- , Rag-/- mice lacking DR3 in all cell types (Rag-/-Dr3-/-), or Rag-/- mice lacking DR3 only on fibroblasts (Rag-/-Dr3∆Col1a2) to induce colitis and fibrosis, assessed by clinical disease activity index, intestinal inflammation, and collagen deposition. Rag-/- mice developed overt colitis with intestinal fibrostenosis. In contrast, Rag-/-Dr3-/- demonstrated decreased inflammation and fibrosis. Despite similar clinical disease and inflammation as Rag-/-, Rag-/-Dr3∆Col1a2 exhibited reduced intestinal fibrosis and attenuated fibroblast activation and migration. RNA-Sequencing of TL1A-stimulated fibroblasts identified Rho signal transduction as a major pathway activated by TL1A and inhibition of this pathway modulated TL1A-mediated fibroblast functions. Thus, direct TL1A signaling on fibroblasts promotes intestinal fibrosis in vivo. These results provide novel insight into profibrotic pathways mediated by TL1A paralleling its pro-inflammatory effects.

Small Molecule Sensors Targeting the Bacterial Cell Wall.

This review highlights recent efforts to detect bacteria using engineered small molecules that are processed and incorporated similarly to their natural counterparts. There are both scientific and clinical justifications for these endeavors. The use of detectable, cell-wall targeted chemical probes has elucidated microbial behavior, with several fluorescent labeling methods in widespread laboratory use. Furthermore, many existing efforts including ours, focus on developing new imaging tools to study infection in clinical practice. The bacterial cell wall, a remarkably rich and complex structure, is an outstanding target for bacteria-specific detection. Several cell wall components are found in bacteria but not mammals, especially peptidoglycan, lipopolysaccharide, and teichoic acids. As this review highlights, the development of laboratory tools for fluorescence microscopy has vastly outstripped related positron emission tomography (PET) or single photon emission computed tomography (SPECT) radiotracer development. However, there is great synergy between these chemical strategies, which both employ mimicry of endogenous substrates to incorporate detectable structures. As the field of bacteria-specific imaging grows, it will be important to understand the mechanisms involved in microbial incorporation of radionuclides. Additionally, we will highlight the clinical challenges motivating this imaging effort.

Sensing Living Bacteria in Vivo Using d-Alanine-Derived 11C Radiotracers.

Incorporation of d-amino acids into peptidoglycan is a unique metabolic feature of bacteria. Since d-amino acids are not metabolic substrates in most mammalian tissues, this difference can be exploited to detect living bacteria in vivo. Given the prevalence of d-alanine in peptidoglycan muropeptides, as well as its role in several antibiotic mechanisms, we targeted this amino acid for positron emission tomography (PET) radiotracer development. d-[3-11C]Alanine and the dipeptide d-[3-11C]alanyl-d-alanine were synthesized via asymmetric alkylation of glycine-derived Schiff-base precursors with [11C]methyl iodide in the presence of a cinchonidinium phase-transfer catalyst. In cell experiments, both tracers showed accumulation by a wide variety of both Gram-positive and Gram-negative pathogens including Staphylococcus aureus and Pseudomonas aeruginosa. In a mouse model of acute bacterial myositis, d-[3-11C]alanine was accumulated by living microorganisms but was not taken up in areas of sterile inflammation. When compared to existing clinical nuclear imaging tools, specifically 2-deoxy-2-[18F]fluoro-d-glucose and a gallium citrate radiotracer, d-alanine showed more bacteria-specific uptake. Decreased d-[3-11C]alanine uptake was also observed in antibiotic-sensitive microbes after antimicrobial therapy, when compared to that in resistant organisms. Finally, prominent uptake of d-[3-11C]alanine uptake was seen in rodent models of discitis-osteomyelitis and P. aeruginosa pneumonia. These data provide strong justification for clinical translation of d-[3-11C]alanine to address a number of important human infections.

Arabinofuranose-derived positron-emission tomography radiotracers for detection of pathogenic microorganisms.

PURPOSE: Detection of bacteria-specific metabolism via positron emission tomography (PET) is an emerging strategy to image human pathogens, with dramatic implications for clinical practice. In silico and in vitro screening tools have recently been applied to this problem, with several monosaccharides including l-arabinose showing rapid accumulation in Escherichia coli and other organisms. Our goal for this study was to evaluate several synthetically viable arabinofuranose-derived 18 F analogs for their incorporation into pathogenic bacteria. PROCEDURES: We synthesized four radiolabeled arabinofuranose-derived sugars: 2-deoxy-2-[18 F]fluoro-arabinofuranoses (d-2-18 F-AF and l-2-18 F-AF) and 5-deoxy-5-[18 F]fluoro-arabinofuranoses (d-5-18 F-AF and l-5-18 F-AF). The arabinofuranoses were synthesized from 18 F- via triflated, peracetylated precursors analogous to the most common radiosynthesis of 2-deoxy-2-[18 F]fluoro-d-glucose ([18 F]FDG). These radiotracers were screened for their uptake into E. coli and Staphylococcus aureus. Subsequently, the sensitivity of d-2-18 F-AF and l-2-18 F-AF to key human pathogens was investigated in vitro. RESULTS: All 18 F radiotracer targets were synthesized in high radiochemical purity. In the screening study, d-2-18 F-AF and l-2-18 F-AF showed greater accumulation in E. coli than in S. aureus. When evaluated in a panel of pathologic microorganisms, both d-2-18 F-AF and l-2-18 F-AF demonstrated sensitivity to most gram-positive and gram-negative bacteria. CONCLUSIONS: Arabinofuranose-derived 18 F PET radiotracers can be synthesized with high radiochemical purity. Our study showed absence of bacterial accumulation for 5-substitued analogs, a finding that may have mechanistic implications for related tracers. Both d-2-18 F-AF and l-2-18 F-AF showed sensitivity to most gram-negative and gram-positive organisms. Future in vivo studies will evaluate the diagnostic accuracy of these radiotracers in animal models of infection.

High Enantiomeric Excess In-Loop Synthesis of d-[methyl-11C]Methionine for Use as a Diagnostic Positron Emission Tomography Radiotracer in Bacterial Infection.

Currently, there exists no accurate, noninvasive clinical imaging method to detect living bacteria in vivo. Our goal is to provide a positron emission tomography (PET) method to image infection by targeting bacteria-specific metabolism. Standard of care methodologies detect morphologic changes, image immunologic response to infection, or employ invasive tissue sampling with associated patient morbidity. These strategies, however, are not specific for living bacteria and are often inadequate to detect bacterial infection during fever workup. As such, there is an unmet clinical need to identify and validate new imaging tools suitable for noninvasive, in vivo (PET) imaging of living bacteria. We have shown that d-[methyl-11C]methionine (d-[11C]Met) can distinguish active bacterial infection from sterile inflammation in a murine infection model and is sensitive to both Gram-positive and Gram-negative bacteria. Here, we report an automated and >99% enantiomeric excess (ee) synthesis of d-[11C]Met from a linear d-homocysteine precursor, a significant improvement over the previously reported synthesis utilizing a d-homocysteine thiolactone hydrochloride precursor with approximately 75-85% ee. Furthermore, we took additional steps toward applying d-[11C]Met to infected patients. d-[11C]Met was subject to a panel of clinically relevant bacterial strains and demonstrated promising sensitivity to these pathogens. Finally, we performed radiation dosimetry in a normal murine cohort to set the stage for translation to humans in the near future.

An Experimental Pipeline for Initial Characterization of Bacterial Type III Secretion System Inhibitor Mode of Action Using Enteropathogenic Yersinia.

Dozens of Gram negative pathogens use one or more type III secretion systems (T3SS) to disarm host defenses or occupy a beneficial niche during infection of a host organism. While the T3SS represents an attractive drug target and dozens of compounds with T3SS inhibitory activity have been identified, few T3SS inhibitors have been validated and mode of action determined. One issue is the lack of standardized orthogonal assays following high throughput screening. Using a training set of commercially available compounds previously shown to possess T3SS inhibitory activity, we demonstrate the utility of an experiment pipeline comprised of six distinct assays to assess the stages of type III secretion impacted: T3SS gene copy number, T3SS gene expression, T3SS basal body and needle assembly, secretion of cargo through the T3SS, and translocation of T3SS effector proteins into host cells. We used enteropathogenic Yersinia as the workhorse T3SS-expressing model organisms for this experimental pipeline, as Yersinia is sensitive to all T3SS inhibitors we tested, including those active against other T3SS-expressing pathogens. We find that this experimental pipeline is capable of rapidly distinguishing between T3SS inhibitors that interrupt the process of type III secretion at different points in T3SS assembly and function. For example, our data suggests that Compound 3, a malic diamide, blocks either activity of the assembled T3SS or alters the structure of the T3SS in a way that blocks T3SS cargo secretion but not antibody recognition of the T3SS needle. In contrast, our data predicts that Compound 4, a haloid-containing sulfonamidobenzamide, disrupts T3SS needle subunit secretion or assembly. Furthermore, we suggest that misregulation of copy number control of the pYV virulence plasmid, which encodes the Yersinia T3SS, should be considered as a possible mode of action for compounds with T3SS inhibitory activity against Yersinia.

Investigation of the Physical and Bioactive Properties of Bromo- and Iodo-Containing Sponge-Derived Compounds Possessing an Oxyphenylethanamine Core.

This research set out to identify compounds from marine sponges that can act as bacterial virulence blockers. Extracts from a total of 80 sponges collected from throughout Indonesia were screened in a high-throughput NF-κB-based screen that identifies compounds capable of inhibiting the bacterial type III secretion system (T3SS) in Yersinia pseudotuberculosis. An extract that was shown to inhibit T3SS-driven NF-κB expression was obtained from an Iotrochota cf. iota sponge and was the source of seven new bromo- and iodo-containing compounds, all of which contain a 2-(4-oxyphenyl)ethan-1-amine core. Five were determined to be new compounds and named enisorines A-E (1-5). The remaining two were determined to be new hemibastadinol analogues named (+)-1-O-methylhemibastadinol 2 (6) and (+)-1-O-methylhemibastadinol 4 (7). All seven compounds inhibited T3SS-dependent YopE secretion and did not affect the growth or metabolic activity of Y. pseudotuberculosis. The most potent inhibitors of T3SS activity were enisorine C (3), enisorine E (5), and (+)-1-O-methylhemibastadinol 2 (6), all of which inhibited YopE secretion by >50% at 30 µM.

Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors.

Antibiotic-resistant bacteria are an emerging threat to global public health. New classes of antibiotics and tools for antimicrobial discovery are urgently needed. Type III secretion systems (T3SS), which are required by dozens of Gram-negative bacteria for virulence but largely absent from nonpathogenic bacteria, are promising virulence blocker targets. The ability of mammalian cells to recognize the presence of a functional T3SS and trigger NF-κB activation provides a rapid and sensitive method for identifying chemical inhibitors of T3SS activity. In this study, we generated a HEK293 stable cell line expressing green fluorescent protein (GFP) driven by a promoter containing NF-κB enhancer elements to serve as a readout of T3SS function. We identified a family of synthetic cyclic peptide-peptoid hybrid molecules (peptomers) that exhibited dose-dependent inhibition of T3SS effector secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa without affecting bacterial growth or motility. Among these inhibitors, EpD-3'N, EpD-1,2N, EpD-1,3'N, EpD-1,2,3'N, and EpD-1,2,4'N exhibited strong inhibitory effects on translocation of the Yersinia YopM effector protein into mammalian cells (>40% translocation inhibition at 7.5 µM) and showed no toxicity to mammalian cells at 240 µM. In addition, EpD-3'N and EpD-1,2,4'N reduced the rounding of HeLa cells caused by the activity of Yersinia effector proteins that target the actin cytoskeleton. In summary, we have discovered a family of novel cyclic peptomers that inhibit the injectisome T3SS but not the flagellar T3SS.

Myeloid ATG16L1 Facilitates Host-Bacteria Interactions in Maintaining Intestinal Homeostasis.

Intact ATG16L1 plays an essential role in Paneth cell function and intestinal homeostasis. However, the functional consequences of ATG16L1 deficiency in myeloid cells, particularly macrophages, are not fully characterized. We generated mice with Atg16l1 deficiency in myeloid and dendritic cells and showed that mice with myeloid Atg16l1 deficiency had exacerbated colitis in two acute and one chronic model of colitis with increased proinflammatory to anti-inflammatory macrophage ratios, production of proinflammatory cytokines, and numbers of IgA-coated intestinal microbes. Mechanistic analyses using primary murine macrophages showed that Atg16l1 deficiency led to increased reactive oxygen species production, impaired mitophagy, reduced microbial killing, impaired processing of MHC class II Ags, and altered intracellular trafficking to the lysosomal compartments. Increased production of reactive oxygen species and reduced microbial killing may be general features of the myeloid compartment, as they were also observed in Atg16l1-deficient primary murine neutrophils. A missense polymorphism (Thr300Ala) in the essential autophagy gene ATG16L1 is associated with Crohn disease (CD). Previous studies showed that this polymorphism leads to enhanced cleavage of ATG16L1 T300A protein and thus reduced autophagy. Similar findings were shown in primary human macrophages from controls and a population of CD patients carrying the Atg16l1 T300A risk variant and who were controlled for NOD2 CD-associated variants. This study revealed that ATG16L1 deficiency led to alterations in macrophage function that contribute to the severity of CD.

Intracellular delivery of fluorescent protein into viable wheat microspores using cationic peptides.

Microspores are specialized generative cells with haploid genome that demonstrate the amenability toward embryogenesis under certain conditions. The induced microspore culture technique is largely exploited by the breeding programs of wheat and other crops due to its high efficiency for generation of the large number of haploid plants in the relatively short period of time. The ability to produce mature double haploid plant from a single cell has also attracted attention of the plant biotechnologists in the past few years. More importantly, the possibility to deliver proteins for improvement of embryogenesis and the genome modification purposes holds great potential for transgene-free wheat biotechnology. In the present study, we examined the ability of cationic and amphipathic cell penetrating peptides (CPPs) to convey a covalently-linked mCherry protein inside the viable microspores. We demonstrate that the affinity of CPPs to the microspore cells dependents on their charge with the highest efficiency of CPP-mCherry binding to the cells achieved by cationic CPPs (penetratin and R9). Additionally, due to overall negative charge of the microspore cell wall, the successful uptake of the protein cargo by live microspore cells is attained by utilization of a reversible disulfide bond between the R9 CPP and mCherry protein. Overall, the approach proposed herein can be applied by the other biotechnology groups for the fast and efficient screening of the different CPP candidates for their ability to deliver proteins inside the viable plant cells.