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Viruses are obligate parasites that rely extensively on host cellular machinery to complete their life cycles. Therefore, examining the fundamental nature behind how viruses interact with their hosts can teach us more about the pathogenesis of these microbes while also contributing to our overall understanding of essential cell biological processes. In a 2021 mSphere of Influence article, the use of live-cell imaging to uncover a unique mechanism of viral assembly was discussed. In this Full Circle review, we highlight the high-resolution imaging techniques currently revolutionizing virology research and discuss how they can be utilized to advance our ability to identify and interrogate novel virus-host interactions.

Katherine Rhodes is a bacteriologist working in the field of host-microbe interaction. In this mSphere of Influence article, she reflects on how two papers, "Spatial ecology of the human tongue dorsum microbiome" by S. Wilbert, J. Mark Welch, and G. Borisy and "Novel peptide from commensal Staphylococcus simulans blocks methicillin-resistant Staphylococcus aureus quorum sensing and protects host skin from damage" by M. Brown et al., impact her research on Neisseria commensalism and host adaptation.

Acinetobacter baumannii is a leading cause of hospital-acquired infections, including ventilator-associated pneumonia. Dietary zinc deficiency is a major risk factor for pneumonia, and hospitalized patients at risk for A. baumannii infection have increased rates of zinc deficiency. We previously showed that dietary zinc deficiency enhanced A. baumannii pneumonia pathogenesis via an IL-13-dependent mechanism in mice. Here, we identified A. baumannii genes required for proliferation in the lungs of zinc-deficient mice using a genome-wide transposon sequencing (Tn-seq) screen. In zinc-deficient mice, Tn insertions in 614 A. baumannii genes led to significant differences in fitness in the lungs at 24 h. Most of these genes were also required in zinc-sufficient control mice. Mutants with disruptions in genes in the purine biosynthetic pathway, such as purI, and acinetobactin iron siderophore pathways were more strongly selected during lung infection in zinc-deficient mice compared to zinc-sufficient mice by Tn-seq. A reconstructed purI mutant was defective compared to wild type during lung infection in zinc-sufficient mice, with the defect further exacerbated in zinc-deficient mice. Thus, A. baumannii purine biosynthesis is required to infect the lung, and its requirement is exacerbated in a zinc-deficient host.IMPORTANCEDietary zinc deficiency is a major risk factor for infection worldwide. In the United States, hospitalized patients are at increased risk of zinc deficiency and A. baumannii pneumonia. In this study, A. baumannii purine biosynthesis was required for lung infection of mice, independent of dietary zinc. Therefore, bacterial purine biosynthesis is an attractive drug target for treating lung infections in patients with variable dietary zinc statuses, such as in hospitalized patients.

The genus Acinetobacter is vast and diverse regarding its hosts. However, it is best known as an opportunistic pathogen that causes hard-to-treat nosocomial infections. Yet, some species of the genus can be beneficial for some hosts. Such is the case of Acinetobacter calcoaceticus, which can have a significant impact on tomato plants, as was recently shown in a paper by Robertson et al. (S. Robertson, A. Mosca, S. Ashraf, A. Corral, et al., mSphere 11:e00842-25, 2026, https://doi.org/10.1128/msphere.00842-25). Importantly, that study also exemplifies how metagenomics in general, but metagenome-assembled genomes in particular, can be employed to understand the functional specialization and identity of the bacterial species dwelling in particular environments.

Multidrug resistance (MDR) of the pathogen Acinetobacter baumannii is a major challenge to global healthcare due to the limited treatment options. The emergence of MDR bacteria necessitates innovative therapeutic approaches, especially given the associated economic burden and the rapid spread of infections. Conventional treatments such as antibiotics and vaccines face significant obstacles. Antimicrobial peptides (AMPs) such as LL37 have potential as an alternative treatment due to their broad-spectrum activity and ability to target specific bacterial structures such as the outer membrane protein A (OmpA). The efficacy of AMPs can be enhanced by using nanobodies (Nbs) that bind to bacterial OmpA, guiding LL37 precisely to its target. In this study, A. baumannii OmpA (AbOmpA)-specific Nbs (NbO7 and NbO13) were efficiently isolated through magnetic-activated cell sorting-based screening of a yeast surface display library, eliminating the need for specialized equipment. Nbs exhibited specific, dose-dependent binding to the target. Conjugation of Nbs with LL37 effectively inhibited the growth of MDR A. baumannii. This approach leverages the natural antimicrobial properties of AMPs and enhances their specificity and effectiveness by targeting bacterial cell surface proteins. LL37-conjugated AbOmpA-Nbs present a promising therapeutic strategy against MDR A. baumannii and other resistant pathogens.IMPORTANCEMultidrug-resistant (MDR) Acinetobacter baumannii poses a major global health threat due to its resistance to nearly all available antibiotics and its persistence in hospital settings. This challenge underscores the urgent need for new therapeutic approaches beyond conventional drugs. In this study, we developed an innovative strategy that combines the human antimicrobial peptide LL37 with nanobodies (Nbs) targeting the outer membrane protein A (OmpA), a key virulence and survival factor of A. baumannii. OmpA-specific Nbs were efficiently isolated from a fully synthetic library using a simple, low-cost selection approach without animal immunization. When conjugated with LL37, these Nbs bound specifically to OmpA and strongly inhibited MDR A. baumannii growth in vitro. Our findings introduce a simple yet powerful platform for generating targeted Nb-peptide conjugates, offering strong potential for adaptation against other antibiotic-resistant pathogens and contributing to the development of next-generation biologics to overcome antibiotic limitations.

Glycosaminoglycans (GAGs), comprising uronic acids and amino sugars, are widely distributed in human tissues such as the intestine and oral cavity. Various bacteria colonize these tissues by assimilating GAGs. During GAG degradation, 4-deoxy-l-threo-5-hexosulose uronate (DHU) is produced. Pectin, an abundant plant component, is also degraded into DHU. DHU is metabolized in a stepwise manner by the isomerase KduI or its nonhomologous isofunctional enzyme DhuI, followed by the reductase KduD or DhuD, belonging to the same reductase-dehydrogenase family. Previous studies have found that the genes encoding isomerase and reductase (kduI-kduD and dhuD-dhuI, respectively) are usually organized in clusters. Therefore, it was believed that the kduI-kduD and dhuD-dhuI clusters evolved independently. However, the discovery of a hybrid kduI-dhuD cluster raised questions regarding the evolution of these clusters. This study investigated the diversity of clusters through a pan-genomic phylogenetic analysis across 3,550 bacterial strains. Among 16 possible cluster structures, 10 types were involved in DHU metabolism. Bacteroidota possessed a hybrid-type kduI-dhuD cluster, while Bacillota, but not Pseudomonadota or Bacteroidota, possessed the cluster dhuD-dhuI. Using public data sets from the human fecal microbiome and environmental habitats, we detected the prevalence of kduI-dhuD and dhuD-dhuI clusters in gut microbes. Although DHU is generated from oligomerized GAG degradation by unsaturated glucuronyl hydrolase (UGL), the UGL gene was frequently found in pathogenic strains containing kduD-kduI, dhuD-dhuI, kduI-dhuD, or dhuD-kduI, indicating that the acquisition of these clusters is advantageous for human colonization.IMPORTANCEGlycosaminoglycans (GAGs), crucial components of the extracellular matrix, play vital roles in host infection by pathogenic bacteria and host colonization by commensal bacteria. The dhuD-dhuI cluster is well conserved within certain phyla, and it appears to have a strong association with GAG metabolism. In contrast, kduI-containing clusters are more widely distributed across bacterial species. Based on the possession ratios of genes encoding the enzymes involved in the production of 4-deoxy-l-threo-5-hexosulose uronate, this study indicates that the substrates differ depending on the specific cluster type.

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.

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.

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.