Inactivation of the Sts enzymes promotes resistance to lethal Staphylococcus aureus infection.

Staphylococcus aureus is a highly infective Gram-positive bacterial pathogen that causes a wide range of diseases in both healthy and immunocompromised individuals. It can evade host immune defenses by expressing numerous virulence factors and toxins. Coupled with the inability of the human host to develop protective immunity against S. aureus, the emergence of antibiotic-resistant strains complicates treatment options. The non-canonical Sts phosphatases negatively regulate signaling pathways in varied immune cell types. To determine the role of the Sts proteins in regulating host responses to a Gram-positive microorganism, we investigated the response of mice lacking Sts expression to S. aureus infection. Herein, we demonstrate that Sts -/- animals are significantly resistant to lethal intravenous doses of S. aureus strain USA300. Resistance is characterized by significantly enhanced survival and accelerated bacterial clearance in multiple peripheral organs. Infected Sts -/- animals do not display increased levels of cytokines TNFα, IFNγ, and IL-6 in the spleen, liver, and kidney during the early stages of the infection, suggesting that a heightened pro-inflammatory response does not underlie the resistance phenotype. In vivo ablation of mononuclear phagocytes compromises the Sts -/- enhanced CFU clearance phenotype. Additionally, Sts -/- bone marrow-derived macrophages demonstrate significantly enhanced restriction of intracellular S. aureus following ex vivo infection. These results reveal the Sts enzymes to be critical regulators of host immunity to a virulent Gram-positive pathogen and identify them as therapeutic targets for optimizing host anti-microbial responses.

ß-Caryophyllene: A Therapeutic Alternative for Intestinal Barrier Dysfunction Caused by Obesity.

Obesity is an excessive accumulation of fat that exacerbates the metabolic and inflammatory processes. Studies associate these processes with conditions and dysregulation in the intestinal tract, increased concentrations of lipopolysaccharides (LPSs) in the blood, differences in the abundance of intestinal microbiota, and the production of secondary metabolites such as short-chain fatty acids. ß-Caryophyllene (BCP) is a natural sesquiterpene with anti-inflammatory properties and with the potential purpose of fighting metabolic diseases. A diet-induced obesity model was performed in 16-week-old C57BL/6 mice administered with BCP [50 mg/kg]. A reduction in the expression of Claudin-1 was observed in the group with a high-fat diet (HFD), which was caused by the administration of BCP; besides BCP, the phylaAkkermansia and Bacteroidetes decreased between the groups with a standard diet (STD) vs. HFD. Nevertheless, the use of BCP in the STD increased the expression of these phyla with respect to fatty acids; a similar effect was observed, in the HFD group that had a decreasing concentration that was restored with the use of BCP. The levels of endotoxemia and serum leptin increased in the HFD group, while in the HFD + BCP group, similar values were found to those of the STD group, attributing the ability to reduce these in conditions of obesity.

Antibody drug conjugates of cleavable amino-benzoyl-maytansinoids.

ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect.

Generational distribution of a Candida glabrata population: Resilient old cells prevail, while younger cells dominate in the vulnerable host.

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.

Pentoxifylline Alone or in Combination with Gentamicin or Vancomycin Inhibits Live Microbe-Induced Proinflammatory Cytokine Production in Human Cord Blood and Cord Blood Monocytes In Vitro.

Neonatal sepsis and its accompanying inflammatory response contribute to substantial morbidity and mortality. Pentoxifylline (PTX), a phosphodiesterase inhibitor which suppresses transcription and production of proinflammatory cytokines, is a candidate adjunctive therapy for newborn sepsis. We hypothesized that PTX decreases live microbe-induced inflammatory cytokine production in newborn blood. Cord blood was stimulated with live microorganisms commonly encountered in newborn sepsis (Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, or Candida albicans) and simultaneously treated with antimicrobial agents (gentamicin, vancomycin, or amphotericin B) and/or clinically relevant concentrations of PTX. Microbial colony counts were enumerated by plating, supernatant cytokines were measured by multiplex assay, intracellular cytokines and signaling molecules were measured by flow cytometry, and mRNA levels were measured by quantitative reverse transcription-PCR. PTX inhibited concentration-dependent E. coli-, S. aureus-, S. epidermidis-, and C. albicans-induced tumor necrosis factor (TNF) and E. coli-induced interleukin-1ß (IL-1ß) production in whole blood, with greater suppression of proinflammatory cytokines in combination with antimicrobial agents. Likewise, PTX suppressed E. coli-induced monocytic TNF and IL-1ß, whereby combined PTX and gentamicin led to significantly greater reduction of TNF and IL-1ß. The anti-inflammatory effect of PTX on microbe-induced proinflammatory cytokine production was accompanied by inhibition of TNF mRNA expression and was achieved without suppressing the production of the anti-inflammatory IL-10. Of note, microbial colony counts in newborn blood were not increased by PTX. Our findings demonstrated that PTX inhibited microbe-induced proinflammatory cytokine production, especially when combined with antimicrobial agents, without enhancing microbial proliferation in human cord blood in vitro, thus supporting its utility as candidate adjunctive agent for newborn sepsis.

Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids.

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.

A Semi-Synthetic Glycoconjugate Vaccine Candidate for Carbapenem-Resistant Klebsiella pneumoniae.

Hospital-acquired infections are an increasingly serious health concern. Infections caused by carpabenem-resistant Klebsiella pneumoniae (CR-Kp) are especially problematic, with a 50 % average survival rate. CR-Kp are isolated from patients with ever greater frequency, 7 % within the EU but 62 % in Greece. At a time when antibiotics are becoming less effective, no vaccines to protect from this severe bacterial infection exist. Herein, we describe the convergent [3+3] synthesis of the hexasaccharide repeating unit from its capsular polysaccharide and related sequences. Immunization with the synthetic hexasaccharide 1 glycoconjugate resulted in high titers of cross-reactive antibodies against CR-Kp CPS in mice and rabbits. Whole-cell ELISA was used to establish the surface staining of CR-Kp strains. The antibodies raised were found to promote phagocytosis. Thus, this semi-synthetic glycoconjugate is a lead for the development of a vaccine against a rapidly progressing, deadly bacterium.

[Non-celiac gluten sensitivity: Another condition that responds to gluten]. / Sensibilidad no celíaca al gluten: Una patología más que responde al gluten.

Remission of gastrointestinal and general symptoms after gluten withdrawal has been described in some non-celiac individuals for nearly 30 years. Only recently, efforts have been made to define this entity, now referred to as "non-celiac gluten sensitivity". It includes patients that clinically respond to gluten free diet without exhibiting allergic or autoimmune features to explain such response. Wheat allergy, celiac disease, irritable bowel syndrome and symptoms induced by high FODMAPs (Fermentable, Oligo-, Di-, Mono-saccharides And Polyols) consumption are the main differential diagnoses. The relationship with neuropsychiatric disorders such as schizophrenia and autism has not been demonstrated, but currently it gives ground to great hope in families with affected children. Epidemiology of non-celiac gluten sensitivity is not clear. It is described as more common among women and less common in children. Genetic and immune factors, changes in intestinal microbiota and non-gluten components present in wheat grains are main factors postulated in the pathogenesis of this condition. To date, there are no specific biomarkers for non-celiac gluten sensitivity and diagnosis is reached by excluding other causes of disease. A trial with gluten-free diet and subsequent gluten challenge is the methodology most frequently used to confirm diagnosis.

[Transition to adulthood of adolescents with inflammatory bowel disease]. / Transición del adolescente con enfermedad inflamatoria intestinal desde la atención pediátrica a la del adulto.

Inflammatory bowel disease (IBD) is a prevalent chronic disorder, often diagnosed during childhood. Studies have suggested that the incidence of IBD in this group of patients is increasing. Children and adolescents with IBD frequently have more extensive and severe disease than adults. Transition is an important concept to ensure optimal health care management of adolescents and young adult patients with chronic physical and medical conditions. During this process there is a change in knowledge, attitudes and behavior towards the disease with a responsibility that gradually shifts from parents to the patient. The success of the transition process depends on the patient, pediatric and adult gastroenterologists. Thus, providers need to understand how to start, maintain and finish this process. When transition process is coordinated, staged and well planned, the adolescent and young adult will acquire the tools needed to successfully self-manage his or her own medical condition. Rather than a universal model of transition, each institution needs to adapt the most efficient model. The aim of this article is to review concepts pertinent to transition management for adolescents and young adults with IBD.

Carbapenem-resistant Klebsiella pneumoniae exhibit variability in capsular polysaccharide and capsule associated virulence traits.

BACKGROUND: Novel therapies are urgently needed to treat carbapenem-resistant Klebsiella pneumoniae (CR-Kp)-mediated infection, which constitute a major health threat in the United States. In order to assess if it is feasible to develop anticapsular antibodies as a potential novel therapy, it is crucial to first systematically characterize capsular polysaccharide (CPS) and virulence traits in these strains. METHODS: Forty CR-Kp were genotyped by pulsed field gel electrophoresis, multilocus sequence typing (MLST), and molecular capsule typing (C-patterns and wzi sequencing). Their biofilm formation, serum resistance, macrophage-mediated killing, and virulence in Galleria mellonella were compared. MAb (1C9) was generated by co-immunization with 2 CPSs, and cross-reactivity was investigated. RESULTS: MLST assigned 80% of CR-Kp isolates to the ST258-clone. Molecular capsule typing identified new C-patterns, including C200/wzi-154, which was widely represented and associated with blaKPC-3-bearing strains. Heterogeneity was detected in biofilm formation and macrophage-mediated killing. Differences in serum resistance correlated with virulence in G. mellonella. ST258 strains carrying blaKPC-3 were less virulent than those with blaKPC-2. MAb 1C9 cross-reacted with 58% of CR-Kp CPSs. CONCLUSIONS: CR-Kp ST258 strains exhibit variability of virulence-associated traits. Differences were associated with the type of KPC gene and CPS. Identification of cross-reacting anti-CPS mAbs encourages their development as adjunctive therapy.

Cleavage of the antitoxin of the parD toxin-antitoxin system is determined by the ClpAP protease and is modulated by the relative ratio of the toxin and the antitoxin.

Differential stability of toxins and antitoxins is the key for the conditional activation and function of Toxin-Antitoxin systems. Here we report the evaluation of the action of cell proteases Lon, ClpAP, ClpXP and ClpYQ on the Kis antitoxin and the Kid toxin of the parD TA system of plasmid R1. In vitro analysis shows that Kis antitoxin, but not the Kid toxin, is cleaved specifically by the ClpAP protease. The Kid toxin is not cleaved either by this protease or by any of the others cell proteases tested but in complex with the Kis antitoxin protects the cleavage of this protein in a way that is dependent on the toxin-antitoxin ratio. We further show that this protection is correlated with the inability of the ClpA chaperone to access the Kis antitoxin when in complex with Kid toxin. The stability of the antitoxin greatly increases in vivo in a clpP- background and plasmid maintenance mediated by the parD system, which is dependent on the differential decay of the antitoxin, is reduced to the levels observed in the absence of a functional toxin. The functional implications of these data are further discussed within the frame of the regulation of the parD system and of the available information on the nature of the toxin-antitoxin complexes formed at different toxin-antitoxin ratios.

Stability of blood lead levels in children with low-level lead absorption.

Current child blood lead (Pb) screening guidelines assume that blood lead levels (BLLs) are relatively stable over time, and that only youngest children are vulnerable to the damaging effects of lower-range BLLs. This study aimed to test the stability of lower-range (≤ 10 µg/dL) child BLLs over time, and whether lower-range BLLs diminished with age among children aged 6 months to 16 years living in a lower-income neighborhood with a density of pre-1986 housing and legacy contamination. Age, sex, family income, age of residence, and/or residence proximity to point sources of Pb, were tested as potential additional factors. Capillary blood samples from 193 children were analyzed by inductively coupled plasma mass spectrometry (ICPMS). Multiple imputation was used to simulate missing data for 3 blood tests for each child. Integrated Growth Curve models with Test Wave as a random effect were used to test BLL variability over time. Among N = 193 children tested, at Time 1 testing, 8.7% had the BLLs ≥ 5 µg/dL (CDC "elevated" BLL reference value at the time of data collection) and 16.8% had BLLs ≥ 3.5 µg/dL (2021 CDC "elevated" BLL reference value). Modeling with time as a random effect showed that the variability of BLLs were attributable to changes within children. Moreover, time was not a significant predictor of child BLLs over 18 months. A sex by age interaction suggested that BLLs diminished with age only among males. Of the additional environmental factors tested, only proximity to a major source of industrial or vehicle exhaust pollution predicted child BLL variability, and was associated with a small, but significant BLL increase (0.22 µg/dL). These findings suggest that one or two BLL tests for only infants or toddlers are insufficient for identifying children with Pb poisoning.

An interdisciplinary team-based approach for significantly reducing lower-level lead poisoning in U.S. children.

Child lead poisoning damages central nervous system, immune, and renal function, and is the longest-standing public health epidemic in U.S. history. While primary prevention is the ultimate goal, secondary intervention is critical for curbing effects among children already exposed. Despite the lowering of child blood lead level (BLL) reference value in 2012 and again in 2021, few changes to secondary intervention approaches have been discussed. This study tested a novel interdisciplinary approach integrating ongoing child BLL-monitoring with education and home mitigation for families living in neighborhoods at high-risk of child lead exposure. In children ages 6 months to 16 years, most of whom had lowest range exposures, we predicted significantly reduced BLLs following intervention. Methods: Twenty-one families with 49 children, were offered enrollment when at least 1 child in the family was found to have a BLL > 2.5 µg/dL. Child BLLs, determined by ICPMS, were monitored at 4- to 6-month intervals. Education was tailored to family needs, reinforced through repeated parent engagement, and was followed by home testing reports with detailed case-specific information and recommendations for no-cost/low-cost mitigation. Results: Ninety percent of enrolled families complied with the mitigation program. In most cases, isolated, simple-to-mitigate lead hazard sources were found. Most prevalent were consumer products, found in 69% (11/16) of homes. Lead paint was identified in 56% (9/16) of homes. Generalized linear regression with Test Wave as a random effect showed that children's BLLs decreased significantly following the intervention despite fluctuations. Conclusion: Lower-level lead poisoning can be reduced through an interdisciplinary approach that combines ongoing child BLL monitoring; repeated, one-on-one parent prevention education; and identification and no-cost/low-cost mitigation of home lead hazards. Biannual child BLL monitoring is essential for detecting and responding to changes in child BLLs, particularly in neighborhoods deemed high-risk for child lead poisoning.

Understanding the Relationship Between Antiviral Prescription Data and COVID-19 Incidence in New York City: A Retrospective Cohort Study.

The coronavirus disease 2019 (COVID-19) pandemic has caused more than 675 million confirmed cases and nearly 7 million deaths worldwide [1]. While testing for COVID-19 was initially centered in health care facilities, with required reporting to health departments, it is increasingly being performed in the home with rapid antigen testing [2]. Most at-home tests are self-interpreted and not reported to a provider or health department, which could lead to delayed reporting or underreporting of cases [3]. As such, there is a strong possibility that reported cases may become a less reliable indicator of transmission over time.

The ribonucleolytic activity of the ribotoxin α-sarcin is not essential for in vitro protein biosynthesis inhibition.

Fungal ribotoxins are toxic secreted ribonucleases that cleave a conserved single phosphodiester bond located at the sarcin/ricin loop of the larger rRNA. This cleavage inactivates ribosomes leading to protein biosynthesis inhibition and cell death. It has been proposed that interactions other than those found at the active site of ribotoxins are needed to explain their exquisite specific activity. The study presented shows the ability of a catalytically inactive α-sarcin mutant (H137Q) to bind eukaryotic ribosomes and interfere with in vitro protein biosynthesis. The results obtained are compatible with previous observations that α-sarcin can promote cell death by a mechanism that is independent of rRNA cleavage, expanding the potential set of activities performed by this family of toxins.

The toxin-antitoxin proteins relBE2Spn of Streptococcus pneumoniae: characterization and association to their DNA target.

The chromosome of the pathogenic Gram-positive bacterium Streptococcus pneumoniae contains between six to 10 operons encoding toxin-antitoxin systems (TAS). TAS are widespread and redundant in bacteria and archaea and their role, albeit still obscure, may be related to important aspects of bacteria lifestyle like response to stress. One of the most abundant TAS is the relBE family, being present in the chromosome of many bacteria and archaea. Because of the high rates of morbility and mortality caused by S. pneumoniae, it has been interesting to gain knowledge on the pneumococcal TAS, among them the RelBE2Spn proteins. Here, we have analyzed the DNA binding capacity of the RelB2Spn antitoxin and the RelB2Spn-RelE2Spn proteins by band-shift assays. Thus, a DNA region encompassing the operator region of the proteins was identified. In addition, we have used analytical ultracentrifugation and native mass spectrometry to measure the oligomerization state of the antitoxin alone and the RelBE2Spn complex in solution bound or unbound to its DNA substrate. Using native mass spectrometry allowed us to unambiguously determine the stoichiometry of the RelB2Spn and of the RelBE2Spn complex alone or associated to its DNA target.

Kis antitoxin couples plasmid R1 replication and parD (kis,kid) maintenance modules.

The coupling between the replication and parD (kis, kid) maintenance modules of R1 has been revisited here by the isolation of a significant collection of conditional replication mutants in the pKN1562 mini-R1 plasmid, and in its derivative, pJLV01, specifically affected in the RNase activity of the Kid toxin. This new analysis aims to identify key factors in this coupling. For this purpose we have quantified and characterized the restriction introduced by parD to isolate conditional replication mutants of this plasmid, a signature of the modular coupling. This restriction depends on the RNase activity of the Kid toxin and it is relieved by either over-expression of the Kis antitoxin or by preventing its degradation by Lon and ClpAP proteases. Based on these data and on the correlation between copy numbers and parD transcriptional levels obtained in the different mutants, it is proposed that a reduction of Kis antitoxin levels in response to inefficient plasmid replication is the key factor for coupling plasmid replication and parD modules.

Effectiveness of COVID-19 Vaccination Mandates and Incentives in Europe.

During 2021-2022 many countries in the European region of the World Health Organization (WHO) adopted mandatory and incentive-based vaccination measures to stimulate immunization against COVID-19. The measures ranged from positive incentive-based programs (i.e., cash incentives, meal discounts, and lotteries) to introducing COVID-19 certificates and enforcing the universal mandatory vaccination with fines. We assessed the effect of such interventions on COVID-19 vaccine uptake in the population of eight countries within the region. An interrupted time series (ITS) analysis was performed using an autoregressive integrated moving average (ARIMA) approach to account for autocorrelation and seasonality. The results showed the immediate positive impact of vaccination incentives on vaccine uptake in most cases, with the highest impact being cash incentives for the population (1197 per million population per day). Discount incentives did not show any significant impact. The introduction of COVID-19 certificates was associated with a significant immediate or gradual increase in daily administered vaccine doses in all the countries included in the study, up to 117,617 doses gained per million per month. The effect of mandatory vaccination for all or some groups of the population varied from a continuous decrease in daily administered doses (332 per million capita per day), no significant effect, or a delayed or temporary increase (1489 per million capita per day).

Serum Antibody Responses against Carbapenem-Resistant Klebsiella pneumoniae in Infected Patients.

Capsular polysaccharide (CPS) heterogeneity within carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strain sequence type 258 (ST258) must be considered when developing CPS-based vaccines. Here, we sought to characterize CPS-specific antibody responses elicited by CR-Kp-infected patients. Plasma and bacterial isolates were collected from 33 hospital patients with positive CR-Kp cultures. Isolate capsules were typed by wzi sequencing. Reactivity and measures of efficacy of patient antibodies were studied against 3 prevalent CR-Kp CPS types (wzi29, wzi154, and wzi50). High IgG titers against wzi154 and wzi50 CPS were documented in 79% of infected patients. Patient-derived (PD) IgGs agglutinated CR-Kp and limited growth better than naive IgG and promoted phagocytosis of strains across the serotype isolated from their donors. Additionally, poly-IgG from wzi50 and wzi154 patients promoted phagocytosis of nonconcordant CR-Kp serotypes. Such effects were lost when poly-IgG was depleted of CPS-specific IgG. Additionally, mice infected with wzi50, wzi154, and wzi29 CR-Kp strains preopsonized with wzi50 patient-derived IgG exhibited lower lung CFU than controls. Depletion of wzi50 antibodies (Abs) reversed this effect in wzi50 and wzi154 infections, whereas wzi154 Ab depletion reduced poly-IgG efficacy against wzi29 CR-Kp We are the first to report cross-reactive properties of CPS-specific Abs from CR-Kp patients through both in vitro and in vivo models.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is a rapidly emerging public health threat that can cause fatal infections in up to 50% of affected patients. Due to its resistance to nearly all antimicrobials, development of alternate therapies like antibodies and vaccines is urgently needed. Capsular polysaccharides constitute important targets, as they are crucial for Klebsiella pneumoniae pathogenesis. Capsular polysaccharides are very diverse and, therefore, studying the host's capsule-type specific antibodies is crucial to develop effective anti-CPS immunotherapies. In this study, we are the first to characterize humoral responses in infected patients against carbapenem-resistant Klebsiella pneumoniae expressing different wzi capsule types. This study is the first to report the efficacy of cross-reactive properties of CPS-specific Abs in both in vitro and in vivo models.

Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE.

Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a approximately 10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.