mBio最新文献

{"title":"Performance of rapid antigen tests to detect SARS-CoV-2 variant diversity and correlation with viral culture positivity: implication for diagnostic development and future public health strategies.","authors":"Heather Goux, Jennetta Green, Andrew Wilson, Shanmuga Sozhamannan, Stephanie A Richard, Rhonda Colombo, David A Lindholm, Milissa U Jones, Brian K Agan, Derek Larson, David L Saunders, Rupal Mody, Jason Cox, Robert Deans, Joseph Walish, Anthony Fries, Mark P Simons, Simon D Pollett, Darci R Smith","doi":"10.1128/mbio.02737-24","DOIUrl":"10.1128/mbio.02737-24","url":null,"abstract":"<p><p>Antigen-based rapid diagnostic tests (Ag-RDTs) provide timely results, are simple to use, and are less expensive than molecular assays. Recent studies suggest that antigen-based testing aligns with virus culture-based results (a proxy of contagiousness at the peak viral phase of illness); however, the performance of Ag-RDTs for newer SARS-CoV-2 variants is unclear. In this study, we (i) assessed the performance of Ag-RDTs and diagnostic antibodies to detect a range of SARS-CoV-2 variants and (ii) determined whether Ag-RDT results correlated with culture positivity. We noted only minor differences in the limit of detection by variant for all assays, and we demonstrated consistent antibody affinity to the N protein among the different variants. We observed moderate to high sensitivity (46.8%-83.9%) for Ag-RDTs when compared to PCR positivity (100%), and all variants were assessed on each assay. Ag-RDT sensitivity and PCR Ct showed an inverse correlation with the detection of viable virus. Collectively, our results demonstrate that commercially available Ag-RDTs offer variable sensitivity compared to PCR, show similar diagnostic validity across variants, and may predict the risk of transmissibility. These findings may be used to support more tailored SARS-CoV-2 isolation strategies, particularly if other studies clarify the direct association between Ag-RDT positivity and transmission risk. The apparent trade-off between sensitivity in the detection of any PCR-positive infection and concordance with infectious virus positivity may also inform new RDT diagnostic development strategies for SARS-CoV-2 and other epidemic respiratory pathogens.</p><p><strong>Importance: </strong>Despite the availability of vaccines, COVID-19 continues to be a major health concern, and antigen-based rapid diagnostic tests (Ag-RDTs) are commonly used as point-of-care or at-home diagnostic tests. In this study, we evaluated the performance of two commercially available Ag-RDTs and a research Ag-RDT to detect multiple SARS-CoV-2 variants using upper respiratory tract swab samples from clinical COVID-19 cases. Furthermore, we determined whether Ag-RDT results correlated with culture positivity, a potential proxy of viral transmissibility. Our results have important implications to inform future testing and response strategies during periods of high COVID-19 transmission with new variants.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0273724"},"PeriodicalIF":5.1,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546309","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolic interplay between <i>Proteus mirabilis</i> and <i>Enterococcus faecalis</i> facilitates polymicrobial biofilm formation and invasive disease.","authors":"Benjamin C Hunt, Vitus Brix, Joseph Vath, Lauren Beryl Guterman, Steven M Taddei, Namrata Deka, Brian S Learman, Aimee L Brauer, Shichen Shen, Jun Qu, Chelsie E Armbruster","doi":"10.1128/mbio.02164-24","DOIUrl":"10.1128/mbio.02164-24","url":null,"abstract":"<p><p>Biofilms play an important role in the development and pathogenesis of catheter-associated urinary tract infection (CAUTI). <i>Proteus mirabilis</i> and <i>Enterococcus faecalis</i> are common CAUTI pathogens that persistently co-colonize the catheterized urinary tract and form biofilms with increased biomass and antibiotic resistance. In this study, we uncover the metabolic interplay that drives biofilm enhancement and examine the contribution to CAUTI severity. Through compositional and proteomic biofilm analyses, we determined that the increase in biofilm biomass stems from an increase in the protein fraction of the polymicrobial biofilm. We further observed an enrichment in proteins associated with ornithine and arginine metabolism in polymicrobial biofilms compared with single-species biofilms. We show that arginine/ornithine antiport by <i>E. faecalis</i> promotes arginine biosynthesis and metabolism in <i>P. mirabilis</i>, ultimately driving the increase in polymicrobial biofilm protein content without affecting viability of either species. We further show that disrupting <i>E. faecalis</i> ornithine antiport alters the metabolic profile of polymicrobial biofilms and prevents enhancement, and this defect was complemented by supplementation with exogenous ornithine. In a murine model of CAUTI, ornithine antiport did not contribute to <i>E. faecalis</i> colonization but was required for the increased incidence of urinary stone formation and bacteremia that occurs during polymicrobial CAUTI with <i>P. mirabilis</i>. Thus, disrupting metabolic interplay between common co-colonizing species may represent a viable strategy for reducing risk of bacteremia.IMPORTANCEChronic infections often involve the formation of antibiotic-resistant biofilm communities that include multiple different microbes, which pose a challenge for effective treatment. In the catheterized urinary tract, potential pathogens persistently co-colonize for long periods of time and the interactions between them can lead to more severe disease outcomes. In this study, we identified the metabolite L-ornithine as a key mediator of disease-enhancing interactions between two common and challenging pathogens, <i>Enterococcus faecalis</i> and <i>Proteus mirabilis</i>. Disrupting ornithine-mediated interactions may therefore represent a strategy to prevent polymicrobial biofilm formation and decrease risk of severe disease.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0216424"},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546305","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"PlsX and PlsY: Additional roles beyond glycerophospholipid synthesis in Gram-negative bacteria.","authors":"Audrey N Rex, Brent W Simpson, Gregory Bokinsky, M Stephen Trent","doi":"10.1128/mbio.02969-24","DOIUrl":"https://doi.org/10.1128/mbio.02969-24","url":null,"abstract":"<p><p>The unique asymmetry of the Gram-negative outer membrane, with glycerophospholipids (GPLs) in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet, works to resist external stressors and prevent the entry of toxic compounds. Thus, GPL and LPS synthesis must be tightly controlled to maintain the integrity of this essential structure. We sought to decipher why organisms like <i>Escherichia coli</i> possess two redundant pathways-PlsB and PlsX/Y-for synthesis of the GPL precursor lysophosphatidic acid (LPA). LPA is then converted by PlsC to the universal precursor for GPL synthesis, phosphatidic acid (PA). PlsB and PlsC are essential in <i>E. coli</i>, indicating they serve as the major pathway for PA synthesis. While loss of PlsX or PlsY individually has little consequence on the cell, the absence of both was lethal. To understand the synthetic lethality of this seemingly redundant PlsX/Y pathway, we performed a suppressor screen. Suppressor analysis indicated that ∆<i>plsXY</i> requires increased levels of glycerol-3-phosphate (G3P), a GPL precursor. In agreement, ∆<i>plsXY</i> required supplementation with G3P for survival. Furthermore, loss of PlsX dysregulated fatty acid synthesis, resulting in increased long-chain fatty acids. We show that although PlsX/Y together contribute to PA synthesis, they also contribute to the regulation of overall membrane biogenesis. Thus, synthetic lethality of ∆<i>plsXY</i> is multifactorial, suggesting that PlsX/Y has been maintained as a redundant system to fine-tune the synthesis of major lipids and promote cell envelope homeostasis.IMPORTANCEGram-negative bacteria must maintain optimal ratios of glycerophospholipids and lipopolysaccharide within the cell envelope for viability. Maintenance of proper outer membrane asymmetry allows for resistance to toxins and antibiotics. Here, we describe additional roles of PlsX and PlsY in <i>Escherichia coli</i> beyond lysophosphatidic acid synthesis, a key precursor of all glycerophospholipids. These findings suggest that PlsX and PlsY also play a larger role in impacting homeostasis of lipid synthesis.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0296924"},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546310","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unprecedented N₂O production by nitrate-ammonifying <i>Geobacteraceae</i> with distinctive N₂O isotopocule signatures.","authors":"Zhenxing Xu, Shohei Hattori, Yoko Masuda, Sakae Toyoda, Keisuke Koba, Pei Yu, Naohiro Yoshida, Zong-Jun Du, Keishi Senoo","doi":"10.1128/mbio.02540-24","DOIUrl":"https://doi.org/10.1128/mbio.02540-24","url":null,"abstract":"<p><p>Dissimilatory nitrate reduction to ammonium (DNRA), driven by nitrate-ammonifying bacteria, is an increasingly appreciated nitrogen-cycling pathway in terrestrial ecosystems. This process reportedly generates nitrous oxide (N₂O), a strong greenhouse gas with ozone-depleting effects. However, it remains poorly understood how N₂O is produced by environmental nitrate-ammonifiers and how to identify DNRA-derived N₂O. In this study, we characterize two novel enzymatic pathways responsible for N₂O production in <i>Geobacteraceae</i> strains, which are predominant nitrate-ammonifying bacteria in paddy soils. The first pathway involves a membrane-bound nitrate reductase (Nar) and a hybrid cluster protein complex (Hcp-Hcr) that catalyzes the conversion of NO₂^- to NO and subsequently to N₂O. The second pathway is observed in Nar-deficient bacteria, where the nitrite reductase (NrfA) generates NO, which is then reduced to N₂O by Hcp-Hcr. These enzyme combinations are prevalent across the domain Bacteria. Moreover, we observe distinctive isotopocule signatures of DNRA-derived N₂O from other established N₂O production pathways, especially through the highest ¹⁵N-site preference (SP) values (43.0‰-49.9‰) reported so far, indicating a robust means for N₂O source partitioning. Our findings demonstrate two novel N₂O production pathways in DNRA that can be isotopically distinguished from other pathways.IMPORTANCEStimulation of DNRA is a promising strategy to improve fertilizer efficiency and reduce N₂O emission in agriculture soils. This process converts water-leachable NO₃^- and NO₂^- into soil-adsorbable NH₄⁺, thereby alleviating nitrogen loss and N₂O emission resulting from denitrification. However, several studies have noted that DNRA can also be a source of N₂O, contributing to global warming. This contribution is often masked by other N₂O generation processes, leading to a limited understanding of DNRA as an N₂O source. Our study reveals two widespread yet overlooked N₂O production pathways in <i>Geobacteraceae</i>, the predominant DNRA bacteria in paddy soils, along with their distinctive isotopocule signatures. These findings offer novel insights into the role of the DNRA bacteria in N₂O production and underscore the significance of N₂O isotopocule signatures in microbial N₂O source tracking.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0254024"},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546317","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of KSHV vaccine platforms and chimeric MHV68-K-K8.1 glycoprotein for evaluating the <i>in vivo</i> immunogenicity and efficacy of KSHV vaccine candidates.","authors":"Wan-Shan Yang, Dokyun Kim, Soowon Kang, Chih-Jen Lai, Inho Cha, Pei-Ching Chang, Jae U Jung","doi":"10.1128/mbio.02913-24","DOIUrl":"https://doi.org/10.1128/mbio.02913-24","url":null,"abstract":"<p><p>Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is an etiological agent of Kaposi's Sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. Considering the high seroprevalence reaching up to 80% in sub-Saharan Africa, an effective vaccine is crucial for preventing KSHV infection. However, vaccine development has been limited due to the lack of an effective animal model that supports KSHV infection. Murine Herpesvirus 68 (MHV68), a natural mouse pathogen persisting lifelong post-infection, presents a promising model for KSHV infection. In this study, we developed KSHV vaccine and a chimeric MHV68 carrying the KSHV glycoprotein, serving as a surrogate challenge virus for testing KSHV vaccines in a mouse model. Among KSHV virion glycoproteins, K8.1 is the most abundant envelope glycoprotein with the highest immunogenicity. We developed two K8.1 vaccines: K8.1 mRNA-lipid nanoparticle (LNP) vaccine and K8.1_26-87-Ferritin (FT) nanoparticle vaccines. Both induced humoral responses in immunized mice, whereas K8.1 mRNA LNP also induced T cell responses. Using BACmid-mediated homologous recombination, the MHV68 M7 (gp150) gene was replaced with KSHV K8.1 gene to generate chimeric MHV68-K-K8.1. MHV68-K-K8.1 established acute and latent infection in the lungs and spleens of infected mice, respectively. Mice immunized with K8.1 mRNA LNP or K8.1_26-87-FT showed a reduction of MHV68-K-K8.1 titer but not MHV68 wild type (WT) titer in the lung. In addition, viral reactivation of MHV68-K-K8.1 was also significantly reduced in K8.1 mRNA LNP-immunized mice. This study demonstrates the effectiveness of two vaccine candidates in providing immunity against KSHV K8.1 and introduces a surrogate MHV68 system for evaluating vaccine efficacy <i>in vivo</i>.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a prevalent virus that establishes lifelong persistent infection in humans and is linked to several malignancies. While antiretroviral therapy has reduced Kaposi's Sarcoma (KS) complications in people with HIV, KS still affects individuals with well-controlled HIV, older men without HIV, and transplant recipients. Despite its significant impact on human health, however, research on KSHV vaccine has been limited, mainly due to the lack of interest and the absence of a suitable animal model. This study addresses these challenges by developing KSHV K8.1 vaccine with two platforms, mRNA lipid nanoparticle (LNP) and FT nanoparticle. Additionally, chimeric virus, MHV68-K-K8.1, was created to evaluate KSHV vaccine efficacy <i>in vivo</i>. Vaccination of K8.1 mRNA LNP or K8.1_26-87-FT significantly reduced MHV68-K-K8.1 titers. Developing an effective KSHV vaccine requires an innovative approach to ensure safety and efficacy, especially for the immunocompromised population and people with limited healthcare resources. This study could be a potential blueprint for future KSHV vaccine development.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0291324"},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546299","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multiple variables influence the finely calibrated antioxidant defenses of <i>Clostridioides difficile</i>.","authors":"Erin B Purcell","doi":"10.1128/mbio.02544-24","DOIUrl":"https://doi.org/10.1128/mbio.02544-24","url":null,"abstract":"<p><p>The obligate anaerobe <i>Clostridioides difficile</i> encodes multiple reductases to detoxify molecular oxygen and reactive oxygen species. Caulat and colleagues have characterized the activity and regulation of four such reductases (L. C. Caulat, A. Lotoux, M. C. Martins, N. Kint, et al., mBio 15:e01591-24, 2024, https://doi.org/10.1128/mbio.01591-24). Each proved critical for clostridial survival in a different range of oxygen concentrations; together, they ameliorate a broad range of oxidative stress levels. Moreover, two previously uncharacterized regulators were found to control reductase gene expression in response to oxidative stress. The genetic repressor Rex and the reductase FdpF are both sensitive to the NAP⁺:NADH ratio, which is affected by a cell's metabolic state as well as redox activity. While oxygen is known to influence the expression of metabolism genes in <i>C. difficile</i>, the mechanisms for cross-talk between the pathways that respond to oxidative and metabolic stress are not well known. The NADH dependence of Rex and FdpF may represent a newly mapped junction between these pathways.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0254424"},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546307","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"On the possibility of yet a third kinetochore system in the protist phylum Euglenozoa.","authors":"Corinna Benz, Maximilian W D Raas, Pragya Tripathi, Drahomíra Faktorová, Eelco C Tromer, Bungo Akiyoshi, Julius Lukeš","doi":"10.1128/mbio.02936-24","DOIUrl":"https://doi.org/10.1128/mbio.02936-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Transmission of genetic material from one generation to the next is a fundamental feature of all living cells. In eukaryotes, a macromolecular complex called the kinetochore plays crucial roles during chromosome segregation by linking chromosomes to spindle microtubules. Little is known about this process in evolutionarily diverse protists. Within the supergroup Discoba, Euglenozoa forms a speciose group of unicellular flagellates-kinetoplastids, euglenids, and diplonemids. Kinetoplastids have an unconventional kinetochore system, while euglenids have subunits that are conserved among most eukaryotes. For diplonemids, a group of extremely diverse and abundant marine flagellates, it remains unclear what kind of kinetochores are present. Here, we employed deep homology detection protocols using profile-versus-profile Hidden Markov Model searches and AlphaFold-based structural comparisons to detect homologies that might have been previously missed. Interestingly, we still could not detect orthologs for most of the kinetoplastid or canonical kinetochore subunits with few exceptions including a putative centromere-specific histone H3 variant (cenH3/CENP-A), the spindle checkpoint protein Mad2, the chromosomal passenger complex members Aurora and INCENP, and broadly conserved proteins like CLK kinase and the meiotic synaptonemal complex proteins SYCP2/3 that also function at kinetoplastid kinetochores. We examined the localization of five candidate kinetochore-associated proteins in the model diplonemid, &lt;i&gt;Paradiplonema papillatum. Pp&lt;/i&gt;CENP-A shows discrete dots in the nucleus, implying that it is likely a kinetochore component. &lt;i&gt;Pp&lt;/i&gt;Mad2, &lt;i&gt;Pp&lt;/i&gt;CLK&lt;sup&gt;KKT10/19&lt;/sup&gt;, &lt;i&gt;Pp&lt;/i&gt;SYCP2L1&lt;sup&gt;KKT17/18&lt;/sup&gt;, and &lt;i&gt;Pp&lt;/i&gt;INCENP reside in the nucleus, but no clear kinetochore localization was observed. Altogether, these results point to the possibility that diplonemids evolved a hitherto unknown type of kinetochore system.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Importance: &lt;/strong&gt;A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplone","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0293624"},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Substrate identification of putative NCS1 and NCS2 nucleobase transporters in <i>Pseudomonas aeruginosa</i>.","authors":"Corey Kennelly, Arthur Prindle","doi":"10.1128/mbio.02434-24","DOIUrl":"https://doi.org/10.1128/mbio.02434-24","url":null,"abstract":"<p><p><i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that can salvage nucleobases from the environment to conserve nutrients that would otherwise be spent on <i>de novo</i> nucleotide biosynthesis. However, little is known regarding the substrate specificity of the 13 putative nucleobase transporters in <i>P. aeruginosa</i>. Here, using a combination of genetic and chemical approaches, we report substrate identifications for 10 putative nucleobase transporters in <i>P. aeruginosa</i>. Specifically, we individually expressed each transporter in a genetic background lacking all 13 putative nucleobase transporters and quantified growth on a panel of 10 nucleobases as sole nitrogen sources. We confirmed these expression-based substrate identifications using targeted genetic knockouts. In a complementary approach, we utilized four toxic nucleobase antimetabolites to characterize antimicrobial activity in these same strains. We identified the sole allantoin transporter as well as transporters for guanine, xanthine, uric acid, cytosine, thymine, uracil, and dihydrouracil. Furthermore, we associated at least five nucleobase transporters with hypoxanthine, which has been recently reported to be an antibiofilm cue in <i>P. aeruginosa</i>. These results provide an initial characterization of the putative nucleobase transporters in <i>P. aeruginosa</i>, significantly advancing our understanding of nucleobase transport in this clinically relevant organism.</p><p><strong>Importance: </strong><i>Pseudomonas aeruginosa</i> is a frequently multidrug-resistant opportunistic pathogen and one of the most common causes of healthcare-acquired infections. While nucleobases are known to support growth in nutrient-limited conditions, recent work showed that adenine and hypoxanthine can also decrease <i>P. aeruginosa</i> biofilm formation by disrupting c-di-GMP metabolism. Thus, nucleobase transport may be relevant to multiple aspects of <i>P. aeruginosa</i> biology and pathogenesis. However, there is currently little known about the transport of nucleobases in <i>P. aeruginosa</i>. Our work reports initial substrate identifications for 10 putative nucleobase transporters in <i>P. aeruginosa</i>, providing new tools to address previously difficult-to-test hypotheses relating to nucleobase transport in this organism.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0243424"},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546311","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Aminoglycoside heteroresistance in <i>Enterobacter cloacae</i> is driven by the cell envelope stress response.","authors":"Ana J Choi, Daniel J Bennison, Esha Kulkarni, Hibah Azar, Haoyu Sun, Hanqi Li, Jonathan Bradshaw, Hui Wen Yeap, Nicholas Lim, Vishwas Mishra, Anna Crespo-Puig, Ewurabena A Mills, Frances Davies, Shiranee Sriskandan, Avinash R Shenoy","doi":"10.1128/mbio.01699-24","DOIUrl":"https://doi.org/10.1128/mbio.01699-24","url":null,"abstract":"&lt;p&gt;&lt;p&gt;&lt;i&gt;Enterobacter cloacae&lt;/i&gt; is a Gram-negative nosocomial pathogen of the ESKAPE (&lt;i&gt;Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas&lt;/i&gt;, and &lt;i&gt;Enterobacter&lt;/i&gt; spp.) priority group with increasing multi-drug resistance via the acquisition of resistance plasmids. However, &lt;i&gt;E. cloacae&lt;/i&gt; can also display forms of antibiotic refractoriness, such as heteroresistance and tolerance. Here, we report that &lt;i&gt;E. cloacae&lt;/i&gt; displays transient heteroresistance to aminoglycosides, which is accompanied with the formation of small colony variants (SCVs) with increased minimum inhibitor concentration (MIC) of gentamicin and other aminoglycosides used in the clinic, but not other antibiotic classes. To explore the underlying mechanisms, we performed RNA sequencing of heteroresistant bacteria, which revealed global gene expression changes and a signature of the CpxRA cell envelope stress response. Deletion of the &lt;i&gt;cpxRA&lt;/i&gt; two-component system abrogated aminoglycoside heteroresistance and SCV formation, pointing to its indispensable role in these processes. The introduction of a constitutively active allele of &lt;i&gt;cpxA&lt;/i&gt; led to high aminoglycoside MICs&lt;i&gt;,&lt;/i&gt; consistent with cell envelope stress response driving these behaviors in &lt;i&gt;E. cloacae&lt;/i&gt;. Cell envelope stress can be caused by environmental cues, including heavy metals. Indeed, bacterial exposure to copper increased gentamicin MIC in the wild-type but not in the Δ&lt;i&gt;cpxRA&lt;/i&gt; mutant. Moreover, copper exposure also elevated the gentamicin MICs of clinical isolates from bloodstream infections, suggesting that CpxRA- and copper-dependent aminoglycoside resistance is broadly conserved in &lt;i&gt;E. cloacae&lt;/i&gt; strains. Altogether, we establish that &lt;i&gt;E. cloacae&lt;/i&gt; relies on transcriptional reprogramming via the envelope stress response pathway for transient resistance to a major class of frontline antibiotic.IMPORTANCE&lt;i&gt;Enterobacter cloacae&lt;/i&gt; is a bacterium that belongs to the WHO high-priority group and an increasing threat worldwide due its multi-drug resistance. &lt;i&gt;E. cloacae&lt;/i&gt; can also display heteroresistance, which has been linked to treatment failure. We report that &lt;i&gt;E. cloacae&lt;/i&gt; shows heteroresistance to aminoglycoside antibiotics. These are important frontline microbicidal drugs used against Gram-negative bacterial infections; therefore, understanding how resistance develops among sensitive strains is important. We show that aminoglycoside resistance is driven by the activation of the cell envelope stress response and transcriptional reprogramming via the CpxRA two-component system. Furthermore, heterologous activation of envelope stress via copper, typically a heavy metal with antimicrobial actions, also increased aminoglycoside MICs of the &lt;i&gt;E. cloacae&lt;/i&gt; type strain and clinical strains isolated from bloodstream infections. Our study suggests aminoglycoside recalcitrance in &lt;i&gt;E. cloacae&lt;/i&gt; could be broadly conserved and cautions against the undesir","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0169924"},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An orphan kinesin in <i>Trypanosoma brucei</i> regulates hook complex assembly and Golgi biogenesis.","authors":"Qing Zhou, Yasuhiro Kurasawa, Huiqing Hu, Thiago Souza Onofre, Ziyin Li","doi":"10.1128/mbio.02634-24","DOIUrl":"10.1128/mbio.02634-24","url":null,"abstract":"<p><p>Kinesins are microtubule-based motor proteins that play diverse cellular functions by regulating microtubule dynamics and intracellular transport in eukaryotes. The early branching kinetoplastid protozoan <i>Trypanosoma brucei</i> has an expanded repertoire of kinetoplastid-specific kinesins and orphan kinesins, many of which have unknown functions. We report here the identification of an orphan kinesin named KIN-G that plays an essential role in maintaining hook complex integrity and promoting Golgi biogenesis in <i>T. brucei</i>. KIN-G localizes to the distal portion of the centrin arm of the flagellum-associated hook complex through association with the centrin arm protein TbCentrin4. Knockdown of KIN-G in <i>T. brucei</i> disrupts the integrity of the hook complex by reducing the length of the centrin arm and eliminating the shank part of the hook complex, thereby impairing flagellum attachment zone elongation and flagellum positioning, which leads to unequal cytokinesis. KIN-G associates with Golgi through a centrin arm-localized Golgi peripheral protein named CAAP1, which maintains Golgi-centrin arm association to facilitate Golgi biogenesis. Knockdown of KIN-G impairs Golgi biogenesis by disrupting CAAP1 at the centrin arm, thereby impairing the maturation of centrin arm-associated Golgi. <i>In vitro</i> microtubule gliding assays demonstrate that KIN-G is a plus end-directed motor protein, and its motor activity is required for hook complex assembly and Golgi biogenesis. Together, these results identify a kinesin motor protein for promoting hook complex assembly and uncover a control mechanism for Golgi biogenesis through KIN-G-mediated maintenance of Golgi-hook complex association.IMPORTANCE<i>Trypanosoma brucei</i> has a motile flagellum, which controls cell motility, cell morphogenesis, cell division, and cell-cell communication, and a set of cytoskeletal structures, including the hook complex and the centrin arm, associates with the flagellum. Despite the essentiality of these flagellum-associated cytoskeletal structures, their mechanistic roles and the function of their associated proteins remain poorly understood. Here, we demonstrate that the orphan kinesin KIN-G functions to promote the biogenesis of the hook complex and the Golgi apparatus. KIN-G exerts this function by mediating the association between centrin arm and Golgi through the centrin arm protein TbCentrin4 and a novel Golgi scaffold protein named CAAP1, thereby bridging the two structures and maintaining their close association to facilitate the assembly of the two structures. These findings uncover the essential involvement of a kinesin motor protein in regulating the biogenesis of the hook complex and the Golgi in trypanosomes.</p>","PeriodicalId":18315,"journal":{"name":"mBio","volume":" ","pages":"e0263424"},"PeriodicalIF":5.1,"publicationDate":"2024-10-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142546296","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}