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The apicoplast is an essential organelle found in Apicomplexa, a large phylum of intracellular eukaryotic pathogens. The apicoplast produces metabolites that are utilized for membrane biogenesis and energy production. A majority of apicoplast-resident proteins are encoded by the nuclear genome and are trafficked to the apicoplast and are referred to as nuclear-encoded and apicoplast-trafficked (NEAT) proteins. In this study, we characterized a NEAT protein named TgBipA, which is a homolog of the highly conserved prokaryotic translational GTPase BipA. BipA is essential for bacterial survival in stress conditions and functions through interactions with the prokaryotic ribosome, although its role is not fully understood. Through genetic knockouts of TgBipA and immunofluorescence imaging, we show that the loss of TgBipA results in apicoplast genome replication defects, disruption of NEAT trafficking, loss of the apicoplast, and ultimately parasite death. Furthermore, we show through comparative studies that this phenotype closely resembles the delayed death phenomenon observed when inhibiting apicoplast translation. Finally, we show that TgBipA is an active GTPase in vitro, and its GTP hydrolysis activity is critical for its cellular function. Our findings demonstrate that TgBipA is a GTPase that has an essential role in apicoplast maintenance, providing new insights into the cellular processes of the organelle.IMPORTANCEToxoplasma gondii, and many other parasites in the phylum Apicomplexa, are pathogens with significant medical and veterinary importance. Most Apicomplexa contain a non-photosynthetic plastid organelle named the apicoplast. This organelle produces essential metabolites, and perturbation of apicoplast function results in parasite death. The apicoplast contains bacterial-like pathways for apicoplast genome replication and expression. Thus, the discovery of the apicoplast leads to optimism that this organelle would provide a wealth of anti-parasitic drug targets. Therefore, the identification and characterization of new apicoplast proteins could provide new opportunities for therapeutic development. In this study, we characterized the function of a protein called TgBipA, a homolog of a highly conserved bacterial GTPase BipA, which has been implicated in the maturation of the 50S ribosomal subunit and adaptation to cellular stress. We show that TgBipA is essential for apicoplast maintenance and parasite survival.

A dysfunctional gut microbiome has become increasingly common in infants born in high-income countries as Bifidobacterium strains no longer dominate the gut microbiome. Probiotics containing Bifidobacterium infantis have been used in breastfed newborns to successfully restore the gut microbiome; however, no studies to date have demonstrated this effect in older breastfed infants whose gut microbiomes are transitioning toward stability and maturity. This is a 9-week randomized controlled trial wherein 2-4 months old exclusively breastfed infants (n = 40) received 0 CFU/day B. infantis EVC001 (placebo), 4.0 × 10⁹ CFU/day B. infantis EVC001 (low), 8.0 × 10⁹ CFU/day B. infantis EVC001 (medium), or 1.8 × 10¹⁰ CFU/day B. infantis EVC001 (high) in equal allocation for 28 consecutive days beginning on day 8. Stool samples were collected on study days 7, 10, 14, 21, 28, 35, 42, and 63. Fecal B. infantis levels were significantly higher in all supplement groups compared with placebo on day 28 and day 63. On day 28, fecal B. infantis levels were significantly higher in infants who received any (low, medium, and high) dose compared with baseline. The abundance of fecal Bifidobacteriaceae significantly increased nearly 2-fold in response to B. infantis EVC001 supplementation. No matter the dose, probiotic supplementation with B. infantis in 2- to 4-month-old exclusively breastfed infants resulted in colonization until at least 1 month post-supplementation.

Importance: This study found that supplementing exclusively breastfed infants with a probiotic, Bifidobacterium infantis EVC001, between 2 and 4 months of age can successfully restore beneficial bacteria in their gut, even after the newborn period. Although previous research showed this effect in newborns, this is the first study to demonstrate that older infants, whose gut microbiomes are typically more stable, can still benefit. The probiotic was effective at all tested doses, with higher levels of B. infantis and overall Bifidobacteriaceae in infants' stool during and even 1 month after supplementation. This study demonstrates that B. infantis can take hold in the gut and potentially improve gut health in older breastfed babies, offering a promising approach to support infant health in settings where beneficial gut bacteria are often missing.

Clinical trials: This study was registered at clinicaltrials.gov as NCT03476447.

The situation regarding drug resistance among gram-negative bacteria is becoming increasingly severe. While antimicrobial peptides are an ideal alternative to traditional antibiotics, single-target natural antimicrobial peptides exhibit limitations, including high toxicity and poor permeability. Given the numerous advantages of dual-target peptides for disease treatment, we designed and synthesized the first membrane/ribosome dual-target antimicrobial peptide, FPON, through a functional peptide splicing strategy utilizing FP-CATH and Oncocin as templates. FPON specifically targets gram-negative bacteria and possesses dual functionalities: the ability to disrupt bacterial membrane integrity and the ability to inhibit protein translation. Additionally, FPON exhibited low toxicity and demonstrated significant activity against drug-resistant bacteria in vitro and in vivo. In conclusion, the results presented in this study provide further evidence that dual-targeted antimicrobial peptides constitute an effective treatment strategy against gram-negative drug-resistant bacteria.IMPORTANCEThe issue of antibiotic drug resistance in gram-negative bacteria is one of grave urgency. While single-target antimicrobial peptides offer a potential solution to antibiotic resistance, therapeutic applications are constrained by their high toxicity and poor penetration. In this study, FP-CATH and Oncocin were used as templates for functional peptide splicing to develop FPON, a novel antimicrobial peptide. FPON was shown to disrupt bacterial membranes and inhibit protein synthesis, effectively eliminating gram-negative bacteria. Moreover, FPON exhibits low toxicity and has a significant effect against drug-resistant bacteria. Our research demonstrates that a dual-target design offers a promising avenue for addressing drug-resistant infections.

Mosquito-borne viruses represent a major global public health threat, with transmission dynamics governed by climatic, ecological, and anthropogenic factors. Yantai City, Shandong Province, situated in a warm-temperate monsoon climate zone, shares geographical and ecological characteristics with regions where mosquito-borne viruses are endemic, creating potential for virus introduction. We used metagenomics to systematically analyze viral communities in mosquitoes from the Yantai region. We collected 8,111 mosquitoes representing four genera and six species, with Culex being predominant (89.8%). High-throughput sequencing revealed 11 viral species spanning 9 families, including Peribunyaviridae and Picornaviridae. Notably, Serbia mononega-like virus 1 and Biggievirus Mos11 represent the first reports from China, with quantitative reverse transcription PCR revealing minimum infection rates of 0.34% and 0.68%, respectively. Phylogenetic analysis revealed close relationships to known viral strains, with several isolates potentially representing novel genera or species. Analysis revealed that Culex quinquefasciatus harbored the greatest viral diversity (five species), with significantly higher viral diversity in agricultural versus urban areas (P < 0.001). Several viruses demonstrated cross-species transmission potential, including Zhee mosquito virus, Zhejiang mosquito virus 3, and Culex tritaeniorhynchus rhabdovirus, all detected across multiple mosquito species. While most viruses appear mosquito-specific, several show close phylogenetic relationships to known pathogens, potentially posing public health risks warranting surveillance. This study addresses knowledge gaps regarding mosquito-borne viruses in the Bohai Rim region and provides a scientific foundation for regional viral surveillance and early warning systems.IMPORTANCEMosquito-borne viruses are a significant global health threat, with the potential to cause widespread disease outbreaks. This study investigated the viral diversity within mosquito populations in Yantai, China, and characterized the molecular features of two emerging RNA viruses. These findings highlight the remarkable viral diversity harbored by Culex mosquitoes and reveal higher viral diversity in agricultural areas compared to urban settings. Several identified viruses exhibit cross-species transmission potential and close phylogenetic relationships to known pathogens, suggesting that they may pose public health risks. Understanding these interactions is essential for predicting how environmental changes may affect virus transmission and the resilience of surveillance and control strategies.

Signal transduction allows bacterial pathogens to sense the host environment and regulate gene expression accordingly for adaptation and survival. While the success of infection largely depends on the timely induction of virulence genes, the activity of the regulatory pathways controlling their expression must be tightly regulated for pathogens to cause disease. Here, we establish that a small RNA (sRNA) promotes the negative feedback control of a master virulence regulator in Salmonella enterica serovar Typhimurium (S. Typhimurium) by repressing a signaling protein essential for its induction in response to an intracellular cue. We show that the virulence regulatory PhoP/PhoQ pathway is inhibited by the PhoP-activated sRNA PinT in mildly acidic pH, an infection-relevant condition encountered by S. Typhimurium inside macrophages. PinT directly represses the translation of ugtL mRNA, which encodes the PhoP activator UgtL. This negative feedback regulation reduces PhoP activity, thereby decreasing the expression of PhoP-activated virulence genes like pagC. PinT-mediated repression of ugtL is predicted to be conserved in Salmonella enterica, but not in the nonpathogenic species Salmonella bongori, thus suggesting that the regulation is relevant for virulence. Our findings uncover how pathogens achieve proper levels of induction of their virulence programs through the post-transcriptional negative feedback regulation of factors enhancing the signaling activity of virulence pathways.

Importance: To cause disease, pathogens must express their virulence genes at the right time and in proper levels. Here, we establish that a small RNA (sRNA) restricts the activation of a regulator critical for the virulence of Salmonella enterica serovar Typhimurium (S. Typhimurium). We show that the sRNA PinT inhibits the activity of the master virulence regulator PhoP by repressing its activator UgtL through a direct interaction with ugtL mRNA. This regulation reduces the expression of PhoP-activated genes. Because PhoP activates PinT and UgtL, the three regulators form a negative feedback loop. That the PinT-mediated repression of ugtL is predicted to occur in Salmonella enterica but not in the nonpathogenic species S. bongori suggests it may be a key virulence determinant. Our results unveil a novel layer of fine-tuning of PhoP activity ensuring that S. Typhimurium induces the proper level of its virulence program in response to an infection-relevant stress condition.

The 5 May 2025 executive order (EO) "Improving the safety and security of biological research" established a federal funding pause for dangerous gain-of-function (DGoF) research, defined as seeking certain experimental outcomes and deemed capable of resulting in significant societal consequences. These moves place institutional biosafety committees central in the identification and self-reporting of DGoF. The previous federal review for research anticipated to result in enhanced potential pandemic pathogens involved a multidisciplinary board, including a bioethicist. From our experience on those boards and based on the EO's mandate to assess the significance of the societal consequences that might result from proposed DGoF research, we suggest a layered review process for the assessment of societal consequences to inform implementation of the EO. In the layered review, proposed research, initially identified based on anticipated experimental outcomes, is confirmed as DGoF through an assessment that is informed by ethical frameworks.

The increase in the infection caused by Acinetobacter baumannii is sustained by the selection of distinct epidemic clonal lineages, which are frequently resistant to a broad range of antimicrobials and possess virulence traits responsible for their persistence in the contaminated environment and spread among patients. The present study aimed to perform an integrated genomic and phenotypic analysis to assess the virulence features of ST25 isolates. A. baumannii isolates assigned to the ST25 epidemic clonal lineage shared high genomic similarity and clustered in four clades (I, II, III, and IV), with clade IV further subdivided into CIVa, CIVb, CIVc, and CIVd. Capsular locus (KL) KL14 was the predominant KL type (47%). Accessory genome analysis showed the presence of tartrate metabolism genes only in CII genomes. CIVb and CIVd ST25 A. baumannii isolates showed higher ability to infect Galleria mellonella larvae than CI, CII, CIII, and CIVc isolates. Hydrogen peroxide resistance was higher in CI, CII, CIVb, and CIVd isolates compared with CIII and CIVc isolates. In desiccation survival tests, CIII, CIVb, and CIVd isolates exhibited prolonged survival. In addition, CI, CII, CIII, CIVb, and CIVd isolates showed higher serum resistance than CIVc isolates. Also, KL14 type and lipooligosaccharide outer core locus (OCL) OCL6 type isolates were significantly more resistant to oxidative stress, to desiccation, and possessed a high ability to kill G. mellonella larvae. A positive and significant correlation was found between AdeB and AdeJ efflux pump expression and hydrogen peroxide resistance.IMPORTANCEIn this study, we characterized the genotypic and phenotypic features of A. baumannii strains assigned to the ST25 epidemic clonal lineage, which were isolated from humans, animals, and the environment. We found that ST25 A. baumannii isolates, irrespective of their antimicrobial resistance, showed peculiar virulence features among clades, isolates assigned to clade IVb and IVd showing the highest virulence and elevated resistance to serum and desiccation. Also, a positive significant correlation was found between the presence of KL14 and outer core locus 6 genotypes and resistance to oxidative stress, resistance to desiccation, and the ability to kill G. mellonella larvae. Phenotypic differences reflected clade identity rather than isolate origin, suggesting that specific virulence traits contribute to the environmental persistence and pathogenic potential of A. baumannii ST25 isolates.

Methicillin-resistant Staphylococcus aureus (MRSA) is a high-priority microorganism that necessitates the development of new treatments, as it causes a substantial disease burden and economic impact globally. MRSA colonizes the skin and anterior nares and can potentially become invasive, leading to pneumonia and soft tissue infection. Additionally, MRSA can establish chronic infections in wounds and medical implants, partly due to its ability to form biofilms. Likewise, the skin commensal Staphylococcus epidermidis also causes similar infections, particularly through its ability to form a plastic-binding biofilm. In this study, we used N-benzyl-N-methyldithiocarbamate (BMDC) in combination with copper or zinc to decrease the viability of MRSA in both planktonic and biofilm settings in vitro, as well as to inhibit biofilm formation by S. epidermidis. We used Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), biomass assessment, colony counts, and metabolism assays to interrogate the state of the bacterium after exposure to metal-BMDC. Furthermore, we compared these metal complexes to the antibiotic vancomycin, one of the current therapeutics used to treat MRSA infections. BMDC enhances copper uptake in bacteria, increasing intracellular copper levels by 70-fold compared to copper alone. Copper intoxication leads to a decrease in metabolic activity that ultimately results in bacterial death. Zinc also combines with BMDC, though likely through a different mechanism, and similarly exerts bactericidal effects. Significantly, both metal-BMDC combinations effectively reduce biofilm formation and eradicate bacteria within established biofilms in vitro, highlighting their potential as promising antimicrobial strategies against MRSA and S. epidermidis biofilms.IMPORTANCEAntimicrobial-resistant bacteria, such as Staphylococcus aureus (MRSA) and Staphylococcus epidermidis, are a significant cause of morbidity and mortality in vulnerable populations, contributing to an escalating health and economic burden. Biofilms are an important reservoir that protects bacteria from immune clearance and antimicrobial agents. However, current strategies to effectively target MRSA biofilms are limited. This research describes a therapeutic approach that can disrupt biofilms in both MRSA and S. epidermidis, thereby enhancing bacterial clearance.

Candida albicans is a normal resident of the human gut and mucosal microbiomes and also an opportunistic fungal pathogen. It undergoes several morphological transitions, one of which is white-opaque switching, where C. albicans reversibly alternates between two distinct cell types, namely, "white" and "opaque." Each state, which is maintained by a complex transcriptional feedback loop, is heritable through many cell divisions. To date, most research works on interactions between C. albicans and the innate immune system have utilized white cells. In this paper, we examine the response of opaque cells following phagocytosis by murine macrophage cell lines and compare it to the response of white cells. White cells are known to rapidly form hyphae that can rupture macrophages, but we show here that opaque cells continue to proliferate as yeast-form opaque cells within the macrophage. Before phagocytosis, white and opaque cells differ markedly in the mRNAs they express and therefore enter macrophages as two distinct types of cells. We were surprised to observe that, within macrophages, the transcriptional profiles of white and opaque cells became much more similar to each other. This convergence was driven, in part, by the upregulation, in white cells, of a set of genes that were already expressed in opaque cells prior to macrophage exposure. These observations indicate that opaque cells, compared to white cells, are "pre-adapted" for life within host macrophages.IMPORTANCEThe human fungal pathogen Candida albicans undergoes several morphological transitions, one of which is white-opaque switching. Although most research works on interactions between C. albicans and the innate immune system have focused on white cells, opaque cells have been shown to interact with macrophages differently compared to white cells. In this study, we examine the transcriptional response of opaque cells to phagocytosis and compare it to that of white cells. Despite differences in how the two cell types proliferate following phagocytosis, their transcriptional responses strongly overlap, and fewer genes are differentially expressed between white and opaque cells following phagocytosis than observed in media lacking macrophages. Unexpectedly, the responses of both white and opaque cells favor genes that were already upregulated in opaque cells (relative to white cells) before exposure to macrophages; these observations suggest that opaque cells are "pre-adapted" for life within macrophages.