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Edwardsiella piscicida is a Gram-negative, intracellular, enteric pathogen that primarily causes hemorrhagic septicemia in fish. It survives and replicates within host cells by delivering a subset of effector proteins via the type III secretion system (T3SS). Previous research has identified a novel T3SS effector, EseQ, in E. piscicida; however, its function remains unclear. This study reveals that EseQ binds to both α-tubulin and GEF-H1 (Rho guanine nucleotide exchange factor 1), causing microtubule destabilization and the release of activated GEF-H1. Active GEF-H1 then stimulates the conversion of GDP-RhoA (the inactive form) to GTP-RhoA (the active form), which subsequently induces stress fiber formation in a ROCK-dependent manner. Stress fibers induced by EseQ alter the architecture of zonula occludens-1-mediated intercellular junctions. This leads to increased permeability of the epithelial barrier, thereby facilitating the translocation of E. piscicida through epithelial cell layers and its invasion into zebrafish larvae. In conclusion, this study demonstrates that the Edwardsiella T3SS effector protein EseQ promotes invasion by manipulating the microtubule and actin cytoskeletons and by disrupting the epithelial barrier.IMPORTANCEEdwardsiella piscicida causes severe hemorrhagic septicemia in marine and freshwater fish worldwide, resulting in significant economic losses for the aquaculture industry (K. Y. Leung, Q. Wang, Z. Yang, and B. A. Siame, Virulence 10:555-567, 2019, https://doi.org/10.1080/21505594.2019.1621648). Our previous research identified a novel type III secretion system effector, EseQ, in E. piscicida whose function remains to be elucidated. In this work, we showed that EseQ binds to tubulin and GEF-H1 and destabilizes microtubules. GEF-H1 released from microtubules activates the RhoA-ROCK-MLCII signaling pathway, leading to stress fiber formation in epithelial cells. EseQ deforms the epithelial barrier and promotes E. piscicida's invasion in a stress fiber-dependent manner. This work contributes to the understanding of the mechanism by which E. piscicida invades host cells.

The 3rd African Microbiome Symposium was held in Cape Town, South Africa, from 20 to 22 November 2024. The symposium featured a diverse range of local and international microbiome research and provided a platform for 79 researchers, students, and industry members to engage in discussions on the microbiome within an African context and focusing on translational research. This meeting review shares highlights, findings, and recommendations derived from the event. Insights from two panel discussions revealed key barriers to microbiome research in Africa, including limited funding, infrastructure gaps, and a shortage of trained local scientists. Recommendations centered on increased investment, institutional training, adherence to ethical guidelines, and the fostering of equitable global partnerships.

Rodent-borne hantaviruses pose a continual public health threat to humans through zoonotic transmission, with case fatality rates of up to 50% in some cases. Human infections can lead to hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome depending on the viral species. Despite the morbidity and mortality associated with this family of viruses, no anti-viral therapeutics or vaccines are available to treat and prevent hantavirus disease. The relative shortage of commercially available reagents to study hantavirus infections in vitro and in vivo likely contributes to the challenges in developing viral countermeasures. This report describes the generation of a panel of mouse monoclonal antibodies that collectively recognize the four viral proteins of Seoul virus (SEOV, Orthohantavirus seoulense), an Old World (OW) hantavirus with worldwide distribution, and the causative agent of HFRS. We have validated the specificity and versatility of these antibodies against a subset of OW and New World hantaviruses in assays relying on antigen recognition in denatured or native conformations. We present several antibodies that specifically recognize the SEOV nucleoprotein and polymerase protein in Western blotting and immunostaining assays. We also identified three novel antibodies directed against the glycoprotein complex that are capable of binding to the N-terminal glycoprotein of all hantaviruses tested. These antibodies are freely available to all hantavirus researchers to add to the small, but growing, collection of reliable and available reagents to be used to study hantavirus biology, identify novel antiviral compounds, and measure viral prevalence in the laboratory and the field.IMPORTANCEPathogenic hantaviruses cause severe hemorrhagic disease and pose a significant public health threat worldwide. Insufficient research into the biology of these viruses has slowed the development of effective direct-acting antivirals and vaccines. Here, we describe the generation and validation of novel, specific monoclonal antibodies for the detection of Seoul virus proteins in vitro. These reagents can be used to fill in critical gaps in knowledge regarding hantavirus entry, protein expression, and particle generation.

The upper respiratory tract (URT) microbiome has emerged as a key component of acute otitis media (AOM) pathophysiology; however, few studies conducted to date have evaluated URT microbiome composition in children with recurrent AOM (rAOM). We collected serial nasopharyngeal samples from a cohort of 58 children, 6 to 35 months of age, over a one-year period. Samples were analyzed using 16S rRNA sequencing and PCR-based assays for common otopathogens and respiratory viruses. Age was strongly associated with differential abundance of specific genera, including increased abundance of genera associated with respiratory health (e.g., Dolosigranulum, Corynebacterium). In contrast, samples collected during AOM episodes or within 30 days of receipt of an antibiotic had a lower relative abundance of these genera. Further, the number of antibiotic-free days prior to sample collection was associated with global changes in microbiome composition. Unsupervised clustering identified three microbiome profiles that differed by incidence of AOM, bacterial otopathogen burden, symptom score, and number of antibiotic-free days prior to sample collection. Increasing age was associated with transition to profiles characterized by lower incidence of AOM and bacterial otopathogen burden, while antibiotic use was associated with transition to a profile associated with greater incidence of AOM. Our findings indicate that alterations of the microbiome associated with aging may contribute to decreased incidence of AOM as children age, while systemic antibiotic use may induce dysbiosis, thereby enhancing AOM susceptibility.

Importance: Ear infections are the most common bacterial infection among young children and the leading cause of healthcare visits and antibiotic prescriptions. This study explores the connection between the microbiome of the nose-the community of microorganisms that live in different areas of the human body-and recurrent ear infections in young children. An analysis of nasal swabs collected from 58 children over a year showed that as children age, they tend to have fewer bacterial pathogens and more species that are associated with a healthy state in their microbiomes. These more mature microbiomes were associated with fewer ear infections. In contrast, recent use of antibiotics was associated with microbiomes that had more bacterial pathogens and that were associated with greater ear infection incidence. Overall, these findings indicate that the microbiome may be a key factor in reduced ear infections as children age.

Enterococci are commensals of the intestinal tract that are intrinsically resistant to cephalosporins, antibiotics that inhibit peptidoglycan synthesis. Prior treatment with cephalosporins is a risk factor for acquiring an enterococcal infection. We previously showed that FtsW, a SEDS (shape, elongation, division, and sporulation) protein, is essential for enterococcal cephalosporin resistance. SEDS proteins catalyze glycosyltransferase reactions to polymerize strands of peptidoglycan. Bacterial genomes typically only encode for two SEDS proteins, FtsW and RodA, that form the core of two different peptidoglycan synthases thought to function at distinct locations in the cell. However, a few bacterial genera, including enterococci, encode homologs of not only FtsW and RodA but also additional SEDS proteins. In general, very little is known about the function of these additional SEDS proteins. The genome of Enterococcus faecalis encodes two additional SEDS homologs, whose expression is induced in response to antibiotic-mediated cell wall stress by the CroS/R two-component system. However, nothing was previously known about the function of these SEDS homologs. In this work, we determined that these two additional SEDS homologs in E. faecalis each possess glycosyltransferase activity in vitro, preferentially associate with distinct bPBPs in E. faecalis, can functionally substitute for either FtsW or RodA (but not both), and are upregulated in a CroR-dependent manner in response to FtsW depletion, enhancing peptidoglycan synthesis and cephalosporin resistance.IMPORTANCESEDS (shape, elongation, division, and sporulation) proteins are transmembrane glycosyltransferases that play a critical role in synthesis of bacterial peptidoglycan. It is well known that most bacteria possess two SEDS protein homologs, known as FtsW and RodA, that participate in peptidoglycan synthesis at distinct locations in the cell. Some bacterial genomes also encode, in addition to FtsW and RodA, additional SEDS protein homologs whose functions are typically poorly characterized. Enterococcus faecalis is a commensal of the human intestinal tract and an important opportunistic pathogen that encodes two such additional SEDS proteins, whose functions have not been reported previously. Our results reveal new insights into the activity and function of these additional SEDS homologs, showing that they are genuine glycosyltransferases that enhance peptidoglycan synthesis and cephalosporin resistance in response to cell wall stress.

Aspergillus fumigatus is a filamentous fungus found in compost and soil that can cause invasive and/or chronic disease in humans. Diagnosis and treatment of aspergillosis often occur when A. fumigatus has formed dense networks of hyphae within the lung. These hyphal networks are multicellular, encased in an extracellular matrix, and have reduced susceptibility to contemporary antifungal drugs, similar to bacterial biofilms. A model of these dense hyphal networks observed in vivo can be recapitulated in vitro using a static, submerged biofilm culture. The mechanisms underlying filamentous fungal cell physiology at different stages of biofilm development remain to be defined. Here, we utilized RNA sequencing, in silico metabolic modeling, and molecular genetics approaches to identify A. fumigatus genes and metabolic pathways critical for biofilm development. These analyses revealed that ethanol and butanediol fermentation pathways are important for the development of a mature A. fumigatus biofilm. Correspondingly, a predicted transcription factor (silG) was observed to be required for mature biofilm development. Taken together, these data define key genes and metabolic pathways critical for A. fumigatus biofilm development.

Importance: Aspergillus fumigatus is the most common etiological agent of a collection of diseases termed aspergillosis. Chronic and invasive manifestations of aspergillosis are highlighted by the development of biofilm-like structures on and in tissue. These biofilm structures are resistant to contemporary antifungal drugs, even for strains that are susceptible by standard antimicrobial susceptibility testing methods. Consequently, understanding the mechanisms by which A. fumigatus induces, develops, and maintains biofilms to evade antifungal therapies is expected to illuminate biofilm-specific therapeutic targets. Here, we identify genes involved in fungal fermentation and regulation of transcription as important mediators of A. fumigatus biofilm development.

The Profession of Microbiology (POM) embodies the bulk of the American Society for Microbiology (ASM) members and represents the career preparation arm of the ASM for academia, industry, and clinical lab professions. The ASM Council on Microbial Sciences hosted a virtual retreat in 2025 to identify the future of the POM. The retreat presentations centered on workforce development, professional development, innovations in technology, and interdisciplinary collaborations. Various aspects were identified, such as the need to prepare for careers in industry, as an important goal of future training. It was also clear that scientists, in all walks of life, need professional development training throughout their careers, from early trainees to senior scientists. Innovations in technology warrant continual training to keep abreast of global issues. Finally, the need for science advocacy and the ability to effectively communicate science to citizens is important. The ASM is best suited to leading the way in the recruitment of young scientists to the field of microbiology and providing the necessary training to keep them ahead of the changing technologies. As such, the ASM is poised to prepare its members for a quickly changing career workplace, one that will require collaboration between the many sciences and the community.

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.