Fatal SARS-CoV-2-Associated Panton-Valentine Leukocidin-producing Staphylococcal Bacteremia: A Nationwide Multicenter Cohort Study.

We reviewed all cases of Panton-Valentine leukocidin-producing Staphylococcus aureus (PVL-SA) bacteremia in Danish children between 2016 and 2021. We found 2 fatal cases with preceding viral prodrome due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given the usual benign course of SARS-CoV-2 infection in children, awareness of possible superinfection with PVL-SA in a child with rapid deterioration is crucial to ensure adequate treatment, including antimicrobial drugs with antitoxin effect.

Skin infections due to Panton-Valentine leukocidin (PVL)-producing S. aureus-Cost effectiveness of outpatient treatment.

INTRODUCTION: Skin and soft tissue infections (SSTI) caused by Panton-Valentine leukocidin (PVL)-producing strains of Staphylococcus aureus (PVL-SA) are associated with recurrent skin abscesses. Secondary prevention, in conjunction with primary treatment of the infection, focuses on topical decolonization. Topical decolonization is a standard procedure in cases of recurrent PVL-SA skin infections and is recommended in international guidelines. However, this outpatient treatment is often not fully reimbursed by health insurance providers, which may interfere with successful PVL-SA decolonization. AIM: Our goal was to estimate the cost effectiveness of outpatient decolonization of patients with recurrent PVL-SA skin infections. We calculated the average cost of treatment for PVL-SA per outpatient decolonization procedure as well as per in-hospital stay. METHODS: The study was conducted between 2014 and 2018 at a German tertiary care university hospital. The cohort analyzed was obtained from the hospital's microbiology laboratory database. Data on medical costs, DRG-based diagnoses, and ICD-10 patient data was obtained from the hospital's financial controlling department. We calculated the average cost of treatment for patients admitted for treatment of PVL-SA induced skin infections. The cost of outpatient treatment is based on the German regulations of drug prices for prescription drugs. RESULTS: We analyzed a total of n = 466 swabs from n = 411 patients with recurrent skin infections suspected of carrying PVL-SA. PVL-SA was detected in 61.3% of all patients included in the study. Of those isolates, 80.6% were methicillin-susceptible, 19.4% methicillin-resistant. 89.8% of all patients were treated as outpatients. In 73.0% of inpatients colonized with PVL-SA the main diagnosis was SSTI. The median length of stay was 5.5 days for inpatients colonized with PVL-SA whose main diagnosis SSTI; the average cost was €2,283. The estimated costs per decolonization procedure in outpatients ranged from €50-€110, depending on the products used. CONCLUSION: Our data shows that outpatient decolonization offers a highly cost-effective secondary prevention strategy, which may prevent costly inpatient treatments. Therefore, health insurance companies should consider providing coverage of outpatient treatment of recurrent PVL-SA skin and soft tissue infections.

Leukotoxin production by Fusobacterium necrophorum strains in relation to severity of liver abscesses in cattle.

Fusobacterium necrophorum, a Gram-negative anaerobe, is the primary etiologic agent of liver abscesses of beef cattle. The bacterium, a member of the microbial community of the rumen, travels to the liver via portal circulation to cause abscesses. The severity of liver abscesses vary from mild with one or two small abscesses to severe with medium to large multiple abscesses. Leukotoxin, a secreted protein, is the critical virulence factor involved in the infection. Our objective was to compare leukotoxin production between strains of F. necrophorum isolated from mild and severe liver abscesses collected from slaughtered cattle. The quantification of leukotoxin was based on assays to measure cytotoxicity and protein antigen concentration. One-hundred strains, 50 from mild and 50 from severe abscesses, were utilized in the study. Cell-free supernatants were prepared from cultures grown in anaerobic broth at 9 and 24 h incubations. The leukotoxic activity was quantified by measuring cytotoxicity based on the release of lactic dehydrogenase from bovine lymphocyte cells, BL3, treated with the culture supernatant. Leukotoxin protein concentration was quantified by a sandwich ELISA assay with a leukotoxin-specific monoclonal antibody as the capture antibody. The leukotoxin activity and concentration were highly variable among the strains within each severity of liver abscesses. Although the leukotoxic activity was unaffected by incubation time, leukotoxin protein concentration was consistently higher at 24 h compared to 9 h incubation. Strains from severe liver abscesses had significantly higher leukotoxic activity and higher protein concentration compared to strains from mild liver abscesses (P < 0.0001) at both 9 and 24 h culture supernatants. Across all strains, the correlation coefficients between leukotoxic activity and leukotoxin concentration at 9 and 24 h were 0.14 (P = 0.17) and 0.47 (P < 0.0001), respectively. In conclusion, strains isolated from severe liver abscesses had significantly higher leukotoxic activities and leukotoxin protein concentrations compared to strains isolated from mild liver abscesses.

Panton-Valentine Leukocidin-Secreting Staphylococcus aureus Pneumonia Complicating COVID-19.

Necrotizing pneumonia induced by Panton-Valentine leukocidin-secreting Staphylococcus aureus is a rare but life-threatening infection that has been described in patients after they had influenza. We report a fatal case of this superinfection in a young adult who had coronavirus disease.

Beyond diphtheria toxin: cytotoxic proteins of Corynebacterium ulcerans and Corynebacterium diphtheriae.

Diphtheria toxin is one of the best investigated bacterial toxins and the major virulence factor of toxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans strains. However, also diphtheria toxin-free strains of these two species can cause severe infections in animals and humans, indicating the presence of additional virulence factors. In this study, we present a first characterization of two proteins with cytotoxic effect in corynebacteria. A putative ribosome-binding protein (AEG80717, CULC809_00177), first annotated in a genome sequencing project of C. ulcerans strain 809, was investigated in detail together with a homologous protein identified in C. diphtheriae strain HC04 (AEX80148, CDHC04_0155) in this study. The corresponding proteins show striking structural similarity to Shiga-like toxins. Interaction of wild-type, mutant and complementation as well as overexpression strains with invertebrate model systems and cell lines were investigated. Depending on the presence of the corresponding genes, detrimental effects were observed in vivo in two invertebrate model systems, Caenorhabditis elegans and Galleria mellonella, and on various animal and human epithelial and macrophage cell lines in vitro. Taken together, our results support the idea that pathogenicity of corynebacteria is a multifactorial process and that new virulence factors may influence the outcome of potentially fatal corynebacterial infections.

Tokiinshi, a traditional Japanese medicine (Kampo), suppresses Panton-Valentine leukocidin production in the methicillin-resistant Staphylococcus aureus USA300 clone.

It is necessary to develop agents other than antimicrobials for the treatment of Staphylococcus aureus infections to prevent the emergence of antimicrobial-resistant strains. Particularly, anti-virulence agents against the Panton-Valentine leukocidin (PVL)-positive methicillin-resistant S. aureus (MRSA), USA300 clone, is desired due to its high pathogenicity. Here, we investigated the potential anti-virulence effect of Tokiinshi, which is a traditional Japanese medicine (Kampo) used for skin diseases, against the USA300 clone. A growth inhibition assay showed that a conventional dose (20 mg/ml) of Tokiinshi has bactericidal effects against the clinical USA300 clones. Notably, the growth inhibition effects of Tokiinshi against S. epidermidis strains, which are the major constituents of the skin microbiome, was a bacteriostatic effect. The data suggested that Tokiinshi is unlikely to affect skin flora of S. epidermidis. Furthermore, PVL production and the expression of its gene were significantly suppressed in the USA300 clone by a lower concentration (5 mg/ml) of Tokiinshi. This did not affect the number of viable bacteria. Moreover, Tokiinshi significantly suppressed the expression of the agrA gene, which regulates PVL gene expression. For the first time, our findings strongly suggest that Tokiinshi has the potential to attenuate the virulence of the USA300 clone by suppressing PVL production via agrA gene suppression.

VqsA controls exotoxin production by directly binding to the promoter of asp in the pathogen Vibrio alginolyticus.

Quorum sensing (QS) system is an important bacterial cell-to-cell signaling system controlling expression of various genes in response to cell densities. In vibrios, LuxR/AphA are two established master QS regulators (MQSRs), and VqsA is recently identified to be the third putative MQSR. As a novel LysR-type regulator, the regulon and the underlying regulation mechanisms of VqsA remains to be elucidated. Here our investigation indicated that the yields of alkaline serine protease (Asp), the exotoxin in Vibrio alginolyticus was dependent on both LuxR and VqsA in growth phase dependent manner. Various in vivo and in vitro analyses including electrophoretic mobility shift assays (EMSA) along with DNase I footprinting investigations demonstrated that VqsA positively controls asp expression through directly binding to the partially palindromic 29 bp binding motif in the promoter region of asp. Moreover, RNA-seq analysis validated the regulatory roles of VqsA in various processes in the organism. Collectively, our data showed that VqsA positively regulates the expression of exotoxin and other virulence-associated genes and is essential for the QS regulation in V. alginolyticus.

Multicenter study of clinical non-ß-lactam-antibiotic susceptible MRSA strains: Genetic lineages and Panton-Valentine leukocidin (PVL) production.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is considered a major cause of healthcare-associated (HA) and community-acquired (CA) infections. Considering non-ß-lactam susceptibility as a potential marker for mecC-MRSA and CA-MRSA, the aim of this study was to determine the frequency and the associated genetic lineages of non-beta-lactam-antibiotic susceptible MRSA (NBLS-MRSA) strains in a multicenter study in Spain. METHODS: A collection of 45 NBLS-MRSA strains recovered in the period from January to June 2016 from 12 Spanish hospitals was analyzed. Molecular typing through spa-type characterization, agr group and multi-locus-sequence typing was performed. Methicillin-resistant genes (mecA and mecC) as well as immune evasion cluster (scn-chp-sak-sea-sep, considering scn gene as the marker of IEC system) and Panton-Valentine leukocidin (PVL) genes were determined with PCR/sequencing. RESULTS: The NBLS-MRSA phenotype was uncommon in the 12 hospitals analyzed (NBLS-MRSA/MRSA frequency: 0.3%-7.7%). All strains contained the mecA gene (and none contained mecC). Twenty-two different spa-types were detected among NBLS-MRSA strains, with spa-t008/agr-I the most prevalent (27%). The main clonal complexes were (CC/%): CC8/42.2%, CC5/33.3% and CC30/4.4%, with ST8 and ST5 as the main sequence types. The PVL toxin was present in 38% of strains (with spa-types t008, t024, t019, t044, t068, t318 and t3060). The IEC genes were detected in 78% of strains: IEC type-B (n=17), type-F (n=16), type-A (n=1) and type-E (n=1); 10 MRSA isolates were scn-negative. CONCLUSION: The NBLS-MRSA phenotype is uncommon in the analyzed hospitals; although no mecC-positive strains were detected, it could be a good marker for MRSA PVL-positive isolates (38%), frequently associated with CA-MRSA infections.

Retrospective study of pneumonia due to Panton-Valentine leukocidin-producing Staphylococcus aureus in Reunion.

OBJECTIVE: Panton-Valentine leukocidin-producing Staphylococcus aureus necrotizing pneumonia is an unusual cause of community-acquired pneumonia, although associated with a high case fatality. This infection mainly affects young individuals, without any history, and is most often preceded by flu-like symptoms. METHOD: We focused on patients presenting with Staphylococcus aureus necrotizing pneumonia in Reunion (Indian Ocean) admitted to the emergency department. We performed a retrospective study based on data collected from laboratory registers and medical files of patients presenting with Staphylococcus aureus necrotizing pneumonia in Reunion between December 2014 and December 2017. RESULTS: A total of 16 patients were recruited for this study, with a median age of 40.5 years. More than half of patients had previously been admitted to the emergency department for acute respiratory distress syndrome or severe sepsis. Fourteen patients were admitted to the intensive care unit and six patients died (five premature deaths). CONCLUSION: Physicians should be aware of this infection during the flu season and quickly adapt the specific antibiotic treatment, including a drug inhibiting toxin production. As methicillin-resistant Staphylococcus aureus is very rarely observed in Reunion, physicians can still adapt the empirical treatment, without glycopeptides.

The effects of iclaprim on exotoxin production in methicillin-resistant and vancomycin-intermediate Staphylococcus aureus.

PURPOSE: Extracellular protein toxins contribute to the pathogenesis of Staphylococcus aureus infections. The present study compared the effects of iclaprim and trimethoprim - two folic acid synthesis inhibitors - with nafcillin and vancomycin on production of Panton-Valentine leukocidin (PVL), alpha haemolysin (AH) and toxic-shock syndrome toxin I (TSST-1) in methicillin-resistant and vancomycin-intermediate S. aureus (MRSA and VISA, respectively). METHODOLOGY: Northern blotting and RT-PCR were used to assess gene transcription; toxin-specific bioassays were used to measure protein toxin production. RESULTS: As shown previously, sub-inhibitory concentrations (sub-MIC) of nafcillin increased and prolonged MRSA toxin gene transcription and enhanced PVL, TSST-1 and AH production. Sub-inhibitory doses of iclaprim and trimethoprim delayed maximal AH gene (hla) transcription and suppressed AH production; both drugs delayed, but neither reduced, maximal TSST-1 production. Trimethoprim significantly increased lukF-PV expression and PVL production compared to both untreated and iclaprim-treated cultures. Higher concentrations of iclaprim and trimethoprim markedly suppressed MRSA growth, mRNA synthesis and toxin production. In VISA, iclaprim, vancomycin and nafcillin variably increased tst and hla expression, but only nafcillin increased toxin production. Despite its ability to increase hla expression, iclaprim was the most potent inhibitor of AH production. CONCLUSIONS: We conclude that, due to its ability to suppress toxin production, iclaprim should be effective against severe staphylococcal infections caused by toxin-producing MRSA and VISA strains, especially given its ability to concentrate at sites of infection such as skin and skin structures and the lung.

Phosphorylation at the D53 but Not the T65 Residue of CovR Determines the Repression of rgg and speB Transcription in emm1- and emm49-Type Group A Streptococci.

CovR/CovS is a two-component regulatory system in group A Streptococcus and primarily acts as a transcriptional repressor. The D53 residue of CovR (CovRD53) is phosphorylated by the sensor kinase CovS, and the phosphorylated CovRD53 protein binds to the intergenic region of rgg-speB to inhibit speB transcription. Nonetheless, the transcription of rgg and speB is suppressed in covS mutants. The T65 residue of CovR is phosphorylated in a CovS-independent manner, and phosphorylation at the D53 and T65 residues of CovR is mutually exclusive. Therefore, how phosphorylation at the D53 and T65 residues of CovR contributes to the regulation of rgg and speB expression was elucidated. The transcription of rgg and speB was suppressed in the strain that cannot phosphorylate the D53 residue of CovR (CovRD53A mutant) but restored to levels similar to those of the wild-type strain in the CovRT65A mutant. Nonetheless, inactivation of the T65 residue phosphorylation in the CovRD53A mutant cannot derepress the rgg and speB transcription, indicating that phosphorylation at the T65 residue of CovR is not required for repressing rgg and speB transcription. Furthermore, trans complementation of the CovRD53A protein in the strain that expresses the phosphorylated CovRD53 resulted in the repression of rgg and speB transcription. Unlike the direct binding of the phosphorylated CovRD53 protein and its inhibition of speB transcription demonstrated previously, the present study showed that inactivation of phosphorylation at the D53 residue of CovR contributes dominantly in suppressing rgg and speB transcription.IMPORTANCE CovR/CovS is a two-component regulatory system in group A Streptococcus (GAS). The D53 residue of CovR is phosphorylated by CovS, and the phosphorylated CovRD53 binds to the rgg-speB intergenic region and acts as the transcriptional repressor. Nonetheless, the transcription of rgg and Rgg-controlled speB is upregulated in the covR mutant but inhibited in the covS mutant. The present study showed that nonphosphorylated CovRD53 protein inhibits rgg and speB transcription in the presence of the phosphorylated CovRD53in vivo, indicating that nonphosphorylated CovRD53 has a dominant role in suppressing rgg transcription. These results reveal the roles of nonphosphorylated CovRD53 in regulating rgg transcription, which could contribute significantly to invasive phenotypes of covS mutants.

Subinhibitory concentrations of tedizolid potently inhibit extracellular toxin production by methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

PURPOSE: Potent extracellular toxins including alpha-haemolysin, Panton-Valentine leukocidin (PVL) and toxic-shock syndrome toxin 1 (TSST-1) significantly contribute to Staphylococcus aureus pathogenesis, thus, toxin suppression is a primary focus in treatment of staphylococcal disease. S. aureus maintains complex strategies to regulate toxin expression and previous data have demonstrated that subinhibitory concentrations of beta-lactam antibiotics can adversely increase S. aureus exotoxin production. The current study evaluates the effects of subinhibitory concentrations of tedizolid, a second-generation oxazolidinone derivative, on expression of staphylococcal exotoxins in both methicillin-resistant and methicillin-sensitive S. aureus. METHODOLOGY: S. aureus exotoxin expression levels were compared at 12 and 24 h following treatment with tedizolid, linezolid, nafcillin or vehicle control. RESULTS: Our findings show that the level of antibiotic required to alter toxin production was strain-dependent and corresponds with the quantity of toxin produced, but both tedizolid and linezolid could effectively reduce expression of alpha-haemolysin, PVL and TSST-1 toxin at subinhibitory concentrations. In contrast, nafcillin showed less attenuation and, in some S. aureus strains, led to an increase in toxin expression. Tedizolid consistently inhibited toxin production at a lower overall drug concentration than comparator agents. CONCLUSION: Together, our data support that tedizolid has the potential to improve outcomes of infection due to its superior ability to inhibit S. aureus growth and attenuate exotoxin production.

Generation of a recombinant Aggregatibacter actinomycetemcomitans RTX toxin in Escherichia coli.

A leukotoxin (LtxA) that is produced by Aggregatibacter actinomycetemcomitans (Aa) is an important virulence determinant in an aggressive form of periodontitis in adolescents. Understanding the function of this protein at the molecular level is critical to elucidating its role in the disease process. To accomplish genetic analysis of the protein structure and relating these observations to toxin function, we have developed an E. coli expression system for the generation and rapid purification of LtxA. Cloning the structural toxin gene, ltxA, from Aa strain JP2 under control of T7 promoter-1 of pCDFDuet-1 vector resulted in expression of a 114 KDa protein which could be easily purified by the presence of a carboxy-terminal engineered double hexahistidine (double-His6) tag and was immunologically reactive with an anti-LtxA monoclonal antibody, but was not cytotoxic. Cloning a second gene, ltxC, an acyltransferase gene, into the vector under control of T7 promoter-2, resulted in expression of the biologically active LtxA. The toxin was extracted from E. coli inclusion bodies, purified by immobilized metal affinity chromatography, and refolded by dialysis. When compared by circular dichroism (CD) spectroscopy analysis, acylated recombinant LtxA has a secondary structure consistent with wt LtxA, while variations in α-helical structure of nonacylated LtxA were observed. No modifications in α-helix were found upon the toxin's binding with liposome-incorporated cholesterol. Our results suggest that pure, biologically active recombinant LtxA can be isolated by a one-step affinity chromatography from E. coli. The toxic and structural properties of the recombinant LtxA are similar to its wt counterpart.

Serious life-threatening multifocal infection in a child, caused by Panton-Valentine leucocidin-producing Staphylococcus aureus (PVL-MSSA).

Groin pain is a frequently occurring complaint in presentations to the Emergency Department. Muscular sprain is often a differential diagnosis, however serious conditions such as pyomyositis should not be ignored. This case report presents a child with atraumatic right groin pain, which was initially diagnosed as a muscular sprain. The patient later re-presented out of hours to the Emergency Department with what was found to be extensive pelvic abscesses. He was subsequently found to have bilateral pneumonia and later developed a pericardial effusion and osteomyelitis of the right iliac bone, sacroiliac joint and sacrum. With multiple surgical interventions and appropriate antibiotics, he made a full recovery and was discharged home after a total admission time of 41 days. The causative organism was found to be Panton-Valentine leucocidin-positive methicillin-susceptible Staphylococcus aureus.

In Vitro generation of Panton-Valentine leukocidin (PVL) in clinical Methicillin-Resistant Staphylococcus aureus (MRSA) and its correlation with PVL variant, clonal complex, infection type.

The amount of Panton-Valentine leukocidin (PVL) is diverse among Staphylococcus aureus isolates from different geographical regions, and its significance in some infections is disputed. However, data concerning this information in China are limited. Fifty-one lukSF-PV+ methicillin-resistant Staphylococcus aureus (MRSA) isolates gathered from varying infections were used for PVL production using enzyme-linked immunosorbent assay, and the quantity was analyzed in correlation with PVL isoform, genetic background of the isolate, and disease category. All isolates generated PVL with a range of 0.43-360.87 µg/mL, of which 56.9% isolates (29/51) generated 51-200 µg/mL of PVL; 11.8% (6/51) yielded PVL more than 200 µg/mL, and the rest (31.4%, 16/51) produced PVL of ≤50 µg/mL. The amount of PVL was not related to its variant and infection type, although isolates from skin and soft tissue infection had relatively high mean and median. Clonal complex (CC) 22 isolates might be the producer of relatively high concentrations of PVL; however, the difference among CCs was not analyzed due to a small number of CC isolates. The relevance of PVL production with the infection type, toxin isoform, and genetic characteristic of isolates may vary by clone type and also needs to be further evaluated using a large sample size and best concentration on in vivo environment.

Remitting infections due to community-acquired Panton-Valentine leukocidin-producing Staphylococcus aureus in the Milan area.

One of the most important Staphylococcus aureus virulence factors is Panton-Valentine leukocidin (PVL). We describe an outbreak of recurrent cutaneous PVL infections in different members of three family clusters. Molecular investigations were performed to confirm the presence of the mecA and PVL genes and to assign the SCCmec type, sequence type (ST) and clonal relatedness. A strain of PVL-producing methicillin-resistant S. aureus (MRSA) was responsible for infection in two related families (A and B), and a third family (C) was infected with PVL-producing methicillin-sensitive S. aureus (MSSA). Molecular investigations revealed the same clone of community-acquired (CA)-MRSA, PVL positive ST8, and SCCmec IV in families A and B and CA-MSSA PVL positive ST15 in family C. S. aureus PVL may give rise to recurrent uncontrolled infections that are difficult to eradicate, and close family contacts are at high risk for transmission.

Epidemiological Trends Observed from Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Blood Cultures at a Japanese University Hospital, 2012-2015.

Despite increasing reports of skin and soft tissue infections caused by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Japan, the extent to which these strains cause nosocomial infections remains unknown, and this is especially true for bloodstream infections. In this study, we molecularly characterized MRSA isolates from Japanese blood samples. Among the 151 MRSA isolates collected from 53 medical facilities in 2011, 115 (76%) and 30 (20%) were classified as staphylococcal cassette chromosome mec (SCCmec) types II and IV, respectively, while the Panton-Valentine leukocidin (PVL) gene was detected in only two isolates. Among 66 MRSA isolates collected from Tokyo Medical University Hospital between 2012 and 2015, 43 (65%) and 20 (30%) were classifiable as SCCmec types II and IV, respectively. In 2015, highly virulent strains, such as the SCCmec type IV/PVL and SCCmec type IV/ toxic shock syndrome toxin-1 clonal types, increased in number. Therefore, the SCCmec type IV clone may cause invasive infections not only in community settings but also in healthcare settings in Japan.

Synthetic Cells Synthesize Therapeutic Proteins inside Tumors.

Synthetic cells, artificial cell-like particles, capable of autonomously synthesizing RNA and proteins based on a DNA template, are emerging platforms for studying cellular functions and for revealing the origins-of-life. Here, it is shown for the first time that artificial lipid-based vesicles, containing the molecular machinery necessary for transcription and translation, can be used to synthesize anticancer proteins inside tumors. The synthetic cells are engineered as stand-alone systems, sourcing nutrients from their biological microenvironment to trigger protein synthesis. When pre-loaded with template DNA, amino acids and energy-supplying molecules, up to 2 × 107 copies of green fluorescent protein are synthesized in each synthetic cell. A variety of proteins, having molecular weights reaching 66 kDa and with diagnostic and therapeutic activities, are synthesized inside the particles. Incubating synthetic cells, encoded to secrete Pseudomonas exotoxin A (PE) with 4T1 breast cancer cells in culture, resulted in killing of most of the malignant cells. In mice bearing 4T1 tumors, histological evaluation of the tumor tissue after a local injection of PE-producing particles indicates robust apoptosis. Synthetic cells are new platforms for synthesizing therapeutic proteins on-demand in diseased tissues.