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The present study investigated T and B cell responses following a second heterologous booster dose of BNT162b2 administered after a two-dose CoronaVac regimen for coronavirus disease 2019 (COVID-19) vaccination in 15 healthcare workers. Blood samples were collected 4 weeks after the first booster and at both 4 and 24 weeks after the second BNT162b2 booster. Interferon-γ-secreting CD4+ and CD8+ T cells were detectable 4 weeks after the first booster, whereas only CD4+ T cells remained detectable at both 4 and 24 weeks after the second booster. Seven of the 15 participants (46.7%) were diagnosed with COVID-19 approximately 16 weeks after receiving the second booster. These individuals exhibited significantly higher frequencies of CD4+ T cells at 24 weeks post-booster than at 4 weeks post-booster. In contrast, the non-COVID-19 group exhibited significantly higher CD4+ T cell responses 4 weeks after the second booster. Memory B cells were detected at low frequencies at all three time points. IgG antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein were detectable at all three time points, with a significant decline observed 24 weeks after the second booster. Overall, CD4+ T and B cell responses induced by a heterologous second booster dose of BNT162b2 following a primary two-dose CoronaVac regimen were rapidly elicited and sustained for at least 6 months.IMPORTANCEThere is limited evidence regarding T and B cell responses following a primary COVID-19 vaccination series with CoronaVac and two heterologous BNT162b2 booster doses. This study investigated the longitudinal T and B cell responses induced by a second heterologous BNT162b2 booster following a primary two-dose CoronaVac COVID-19 vaccination regimen. These results demonstrate that CD4+ T cells induced by the second heterologous BNT162b2 booster play a key role in protection against SARS-CoV-2 infection and progression to severe disease. This study suggests the need for the future consideration of repeated emergency vaccine-boosting strategies in response to emerging viral infections.

Bacterial populations often display remarkable morphological heterogeneity. Fluorescence-activated cell sorting (FACS) is an important tool for understanding this diversity. FACs allows researchers to obtain pure samples of each morphological variant (or morphotype) that is present within a mixed population of cells and thus permits each morphotype to be phenotyped. In FACS, cells are first labeled with fluorescent markers, such as antibodies or transgenic constructs, and then separated out based on their possession of these labels. However, since the development of fluorescent labels requires a priori knowledge of bacterial biology, it is often impossible to apply FACS to understudied and/or unculturable bacteria. This challenge has limited our capacity to investigate the biology of bacterial size and shape in all but a small, largely culturable subset of bacterial taxa. Here, we present an innovative strategy that permits label-free cell sorting of bacterial morphotypes, using an unculturable, pleiomorphic pathogen (Pasteuria ramosa) as a model bacterium. We show that imaging flow cytometry (IFC) can be used to systematically identify light-scattering and autofluorescence "signatures" of bacterial morphotypes, on which basis cell sorting can be conducted. Critically, our IFC-enabled cell sorting strategy yields samples of sufficient purity (>90%) for common downstream analyses, for example, "-omics" analyses. Our work represents an innovative application of IFC and provides an economical, widely applicable solution to a central problem in the study of bacterial diversity.IMPORTANCEBacteria come in many different shapes and sizes. Why this morphological variation exists is a long-standing question in microbiology, but it remains difficult to answer. To phenotype different morphological variants (morphotypes) within a bacterial population, we need to separate them from one another. This is normally achieved using fluorescence-activated cell sorting, whereby morphotypes are labeled with fluorescent antibodies and separated on the basis of their differential fluorescence. Unfortunately, it is difficult to develop fluorescent labels specific to unculturable or poorly studied bacteria because of the limited availability of appropriate molecular tools. Here, we demonstrate that imaging flow cytometry can be used to design and validate label-free cell sorting strategies. Recently, there has been a resurgence of interest in bacterial morphological diversity and a call to expand its study across the tree of life. Our work will help microbiologists to answer this call.

Tuberculosis (TB) is one of the most common infectious diseases caused by bacteria worldwide. The increasing prevalence of multidrug-resistant TB (MDR-TB) and latent TB infection (LTBI) has intensified the global TB burden. Therefore, the development of new drugs for MDR-TB and LTBI is urgently required. We have reported that the derivative of dithiocarbamate sugar derivative, 2-acetamido-2-deoxy-β-D-glucopyranosyl N,N-dimethyldithiocarbamate (OCT313), exhibits anti-mycobacterial activity against MDR-MTB. Here, we identified the target of OCT313. In experimentally generated OCT313-resistant bacteria, adenine at position 1,092 in the metabolic enzyme phosphotransacetylase (PTA) gene was replaced with cytosine. This mutation is a nonsynonymous mutation that converts methionine to leucine at position 365 in the PTA protein. OCT313 inhibited the enzymatic activity of recombinant wild-type PTA, but not of the mutant PTA (M365L). PTA is an enzyme that produces acetyl-coenzyme A (acetyl-CoA) from acetyl phosphate and CoA and is involved in metabolic pathways; therefore, it was expected to also be active against dormant Mycobacterium tuberculosis bacilli. OCT313 exhibits antibacterial activity in the Wayne model of dormancy using Mycobacterium bovis BCG, and overexpression of PTA in OCT313-resistant bacilli restored sensitivity to OCT313. Collectively, the target of OCT313 is PTA, and OCT313 is a promising antimicrobial candidate for MDR-TB and LTBI.IMPORTANCEThrough this study, we propose a new target for the development of medicines to treat multidrug-resistant tuberculosis and latent tuberculosis infection. The target enzyme phosphotransacetylase (PTA) is a key enzyme that functions in major metabolic pathways, and the homologous structures of PTA enzymes vary greatly among bacterial species. Since the treatment of mycobacterial disease is long term, the development of antibiotics targeting PTA is useful for species-specific therapy.

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.

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.

One exciting class of future genetic devices could be those deployed in microbes that join complex microbial environments in the wild. We sought to determine whether genetic parts designed for monoculture are predictable when used in co-culture by testing constitutive Anderson promoters driving the expression of chromoproteins from a plasmid. In Escherichia coli monoculture, a high copy number origin of replication causes stochastic expression regardless of promoter strength, and high constitutive Anderson promoter strength leads to selection for inactivating mutations, resulting in inconsistent chromoprotein expression. Medium- and low-strength constitutive Anderson promoters function more predictably in E. coli monoculture but experience an increase in inactivating mutations when grown in co-culture over many generations with Pseudomonas aeruginosa. Expression from regulated promoters instead of constitutive Anderson promoters can lead to stable expression in a complex wastewater culture. Overall, we show intraspecies selection for inactivating mutations due to a competitive growth advantage for E. coli that do not express the genetic device compared to their peers that retain the functional device. We show additional interspecies selection against the functional device when E. coli is co-cultured with another organism. Together, these two selection pressures create a significant barrier to genetic device function in microbial communities that we overcome by utilizing a regulated E. coli promoter. Future strategies for genetic device design in microorganisms that need to function in a complex microbial environment should focus on regulated promoters and/or strategies that give the microorganism carrying the device a selective or growth advantage.

Importance: First-generation biotechnology focused on genetic devices designed for use in monoculture conditions. One class of next-generation biotechnology devices could be designed to function in complex ecosystems with other organisms, so we sought to create conditions where the genetic device retained function when the organism carrying it is in co-culture with other organisms. We discovered that when the genetic device is a significant resource burden on the organism carrying the device, mutations will be selected for due to intraspecies and interspecies selection pressures, and the device will be rendered non-functional. Therefore, genetic device design for complex ecosystems in next-generation biotechnology needs to balance functionality of the genetic device with the need to reduce resource burden on the organism carrying it.

Intrastrain genetic and phenotypic heterogeneity of Pseudomonas aeruginosa is a hallmark of chronic lung infections in individuals with cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Although the coexistence of multiple P. aeruginosa lineages within a single host is well documented, the impact of this heterogeneity on infection microbiogeography remains poorly understood. We previously showed that loss of the lipopolysaccharide (LPS) O-specific antigen (OSA) alters P. aeruginosa aggregate assembly. Since OSA-deficient variants are common in chronic pulmonary infections and associated with increased pathogenesis and immune evasion, we investigated whether intrastrain OSA diversity shapes infection microbiogeography. We constructed mixed populations containing equal ratios of OSA-deficient variants and wild-type (WT) cells and examined aggregate assembly and population structures in a synthetic CF sputum model (SCFM2). To assess OSA heterogeneity in vivo, we used a murine pneumonia model combined with hybridization chain reaction (HCR) RNA-FISH and whole-tissue clearing to visualize spatial organization in the airways. In SCFM2, OSA-deficient variants increased total population size, reduced WT aggregate size, and altered spatial organization. We employed 2-plex HCR RNA-FISH to distinguish WT and OSA-deficient variants in murine lungs. Interestingly, in contrast to in vitro conditions, OSA-deficient cells led to significantly larger WT aggregates in the airways. These findings highlight the role of intrastrain genetic heterogeneity in shaping infection microbiogeography and provide a framework for understanding how population dynamics influence microbial physiology and host-pathogen interactions at the micron scale.IMPORTANCEIntrastrain genetic and phenotypic diversity within Pseudomonas aeruginosa populations is common in chronic pulmonary infections. While this intrastrain heterogeneity is a hallmark of chronic infection, its consequences for the spatial organization of P. aeruginosa within the airways remain unclear. Here, we demonstrate that the loss of O-specific antigen in a subpopulation of P. aeruginosa significantly alters the spatial architecture of P. aeruginosa, without changing the total population size or composition. Using a combination of tissue clearing and hybridization chain reaction RNA-FISH in a murine lung infection model, we mapped the localization of genetically distinct P. aeruginosa variants in mixed populations in vivo. These findings reveal that genetic diversification within a strain can reshape the infection landscape at the micron scale, highlighting the overlooked role of intrastrain dynamics in shaping the microbiogeography of infections and influencing host-pathogen interactions.

When susceptible bacterial cultures are treated with antibiotics, some cells can survive treatment without heritable resistance, giving rise to susceptible daughter cells in a phenomenon termed antibiotic persistence. Current models of fluoroquinolone (FQ) persistence in stationary-phase cultures posit that post-treatment resuscitation is dependent on double-stranded break (DSB) repair through RecA-mediated homology-directed repair. Previously, we reported that stationary-phase P. aeruginosa does not depend on RecA to persist. In this work, we ask whether P. aeruginosa FQ persisters from stationary-phase cultures suffer DSBs at all. We measured DSB formation in Levofloxacin (LVX)-treated cells recovering from treatment using strains expressing fluorescently labeled DSB-binding protein, Gam. We find that, surprisingly, the majority of P. aeruginosa LVX persisters survive treatment without apparent DSBs. Persisters that have evidence of DSBs take longer until their first division compared to persisters without DSBs. Additionally, the fates of their progenies suggest that persisters may cope with DSBs by repair or damage sequestration. These observations pave the way for mechanistic studies into P. aeruginosa FQ persistence and highlight the need for single-cell tools to track FQ-induced damage.

Importance: Pseudomonas aeruginosa is an opportunistic pathogen of significant clinical interest. When susceptible cultures of P. aeruginosa are treated with fluoroquinolone (FQ) antibiotics, some cells survive treatment and regrow in a phenomenon termed antibiotic persistence. Studies in Escherichia coli and other bacterial species suggest that FQ persisters survive by repairing DNA double-stranded breaks (DSBs) after antibiotic removal. In this study, we show that most stationary-phase P. aeruginosa survive by avoiding DSBs rather than repairing them.

DNA ligases are a fundamental class of enzymes required for DNA replication and repair. They catalyze the formation of phosphodiester bonds, specifically at single-strand breaks in double-stranded DNA. The nuclear genome of malaria parasites encodes a single DNA ligase that is likely involved in nuclear and organellar DNA replication and repair. DNA ligase I from Plasmodium falciparum (PfLig1) has been biochemically characterized and shown to possess nick-sealing activity. However, its localization and function in the three genome-containing compartments-the nucleus, apicoplast, and mitochondrion-of the malaria parasites remain unknown. Here, we found that Lig1 is located primarily in the nucleus in both human and rodent malaria parasites throughout the parasite life cycle. Furthermore, we detected its presence in organelles via a chromatin immunoprecipitation-PCR assay. Our attempts to disrupt Plasmodium berghei Lig1 (PbLig1) in the blood stages have failed, indicating that the gene is likely essential. Next, we used an Flp/FRT-based conditional mutagenesis system that silences gene function in sporozoites. We demonstrated that PbLig1 is essential for parasite liver-stage development. Sporozoites lacking PbLig1 invade hepatocytes but arrest growth during mid-liver-stage development. PbLig1 cKO parasites undergo limited nuclear division and present a reduced DNA content that fails to increase beyond mid-liver stage of development. These data suggest that Lig1 is an essential enzyme for parasite blood- and liver-stage development.IMPORTANCEUnlike mammalian cells that possess multiple DNA ligases, the malaria parasite's nuclear genome encodes a single DNA ligase. This single DNA ligase is likely involved in both DNA replication and DNA repair. However, the importance of parasite DNA ligase remains largely unknown. Here, we show that Plasmodium Lig1 is primarily found within the nucleus, but it also exhibits a distribution across parasite organelles. Knockout of PbLig1 in sporozoites abolishes parasite liver-stage development, preventing the formation of hepatic merozoites and ultimately blocking the transition from the liver to the blood stage of infection. More specifically, PbLig1 is essential for nuclear division during hepatic schizogony. These findings enhance our understanding of the role of DNA ligase I in malaria parasite liver-stage development.