mBio

Triazole antifungals inhibit the lanosterol C14-demethylase Cyp51A/B and are the first-line treatment for invasive aspergillosis caused by Aspergillus fumigatus. We previously proposed that triazoles impose a secondary constraint on ergosterol biosynthesis through negative feedback regulation of HMG-CoA reductase (Hmg1) through its sterol sensing domain (SSD), and SSD mutations compromise this regulation to produce resistance. The molecular mechanism underpinning this feedback remained unknown. In this study, we show that voriconazole drives accelerated protein degradation of Hmg1 and that SSD mutation abolishes this regulated degradation. Triazole-induced Hmg1 degradation is proteasome-dependent and partially requires the ERAD E3 ubiquitin ligase, HrdA, and INSIG ortholog, InsA, both conserved regulators of HMG-CoA Reductase abundance. We provide genetic evidence that the initiating negative feedback signal is not triazole acting directly on Hmg1 but lanosterol accumulation resulting from Cyp51A/B inhibition. To test whether depleting Hmg1 protein could re-sensitize resistant strains, we utilized a GFP-targeted degradation system designed to target Hmg1 for degradation without engaging its SSD. Combining this system with voriconazole treatment reduced the MIC at least four-fold in both conidia and established hyphae in susceptible and resistant strains. Together, our findings support a model wherein triazole-induced lanosterol accumulation drives HrdA/InsA-dependent accelerated degradation of Hmg1 and establish that pharmacologically inducing this secondary mechanism can enhance triazole activity in both susceptible and resistant isolates.

Although Burkholderia cenocepacia exhibits biosurfactant-dependent phenotypes and possesses an rhl operon, the chemical identity of its surface-active product has long remained unresolved. The rhl operon of B. cenocepacia harbors an additional gene, rhlM, predicted to encode a methyltransferase, and recently linked to methylated rhamnolipid synthesis in a few Burkholderiaceae. Here, we hypothesized that this biosurfactant remained cryptic due to low expression under standard laboratory conditions. Using a chromosomal Prhl-luxCDABE fusion reporter, we screened diverse nutritional conditions and identified a defined medium containing galactose and urea that strongly induced rhl transcription. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of B. cenocepacia K56-2 extracts identified the biosurfactants as methylated dirhamnolipid congeners, predominantly L-rhamnosyl-L-rhamnosyl-3-hydroxytetradecanoyl-3-hydroxytetradecanoate methyl ester (RR-C14-C14Me). Extending our analysis across the Burkholderia cepacia complex (Bcc) revealed that methylated rhamnolipid production is widespread and restricted to rhl-positive species. Comparative genomic and protein structural analyses further demonstrated high conservation of both the rhlM-containing operon architecture and the RhlM protein across the Bcc. Together, our findings identify the long-standing cryptic biosurfactant of B. cenocepacia as methylated rhamnolipids and establish rhamnolipid methylation as a conserved hallmark across the Bcc, shifting the paradigm of rhamnolipid structural diversity from an isolated exception to a widespread rule.

Haemophilus influenzae is a Gram-negative bacterium that causes pneumonia, otitis media, and invasive infections such as meningitis and bacteremia. A vaccine that confers protection against disease caused by H. influenzae serotype b is currently available and is highly effective, but infections caused by non-b serotypes and non-typeable strains are still prevalent and increasing, suggesting a need for a broadly cross-protective vaccine that will protect against infection by all H. influenzae strains. To address the need for broad cross-protection, this study focused on the surface lipoprotein component of the bipartite bacterial transferrin receptor, transferrin binding protein B (TbpB), which is universally present H. influenzae, irrespective of encapsulation status, and is essential for bacterial colonization and pathogenesis. To prevent vaccine escape, we assessed the sequence and structural diversity among TbpB variants derived from local and international H. influenzae isolates and observed that these sequences cluster independently of encapsulation status, suggesting that conferring protection against all H. influenzae strains regardless of capsule type or presence of a capsule using a TbpB-based vaccine is feasible. In addition, our diversity analysis also revealed that the C-terminal lobe (C-lobe) of TbpB contains several large, highly variable loops. After immunizing mice with a trivalent vaccine consisting of a representative set of TbpB variants, we found that the resulting antiserum demonstrated modest cross-reactivity against heterologous TbpB variants. However, immunizing with a loopless C-lobe (LCL) that lacks the large, highly variable loops we had identified elicited broad cross-reactivity against a panel of intact TbpB variants. This suggests that while the intact TbpB may only generate a moderately cross-reactive antibody response, a vaccine consisting of a single LCL may be able to elicit a sufficiently cross-reactive humoral response against diverse TbpB variants, and in turn, confer broad cross-protection targeting both encapsulated and non-typeable H. influenzae.

The emergence of antimicrobial resistance restricts the clinical efficacy of therapeutic antibiotic drugs. Elucidating the molecular mechanisms by which bacteria evade antibiotic activities will lead to novel therapeutic approaches. Therefore, it is critical to understand the mechanisms that govern susceptibility to antibiotics. Fluoroquinolones are a class of antibiotics widely used to treat both Gram-positive and Gram-negative bacterial infections. Resistance to fluoroquinolones is widespread in multiple pathogenic bacteria, limiting their therapeutic usefulness. Resistance to fluoroquinolones is well-known to be mediated by Point mutations in the genes encoding the cellular targets of fluoroquinolones, type-II topoisomerases, leads to clinically relevant resistance. However, several recent studies have revealed that dysregulation of translation influences fitness during fluoroquinolone exposure. For example, several screens have identified insertions in gene that encodes elongation factor P (EF-P) increases survival to fluoroquinolones. Here, we sought to understand the impact of EF-P on fluoroquinolone survival using the Gram-positive model organism Bacillus subtilis. We found that loss of EF-P increases the survival of B. subtilis exposed to fluoroquinolones by several orders of magnitude. Furthermore, we demonstrate that fluoroquinolone sensitivity is dependent on post-translational modification of EF-P, indicating that it is dependent on EF-P activity. Remarkably, loss of EF-P increases survival of cells lacking RecA, a key DNA repair protein, which is crucial for the survival of fluoroquinolone drugs. Using transcriptomics, we identify key cellular response pathways differentially regulated in cells lacking EF-P during fluoroquinolone treatment. We conclude that EF-P regulates diverse mechanisms that drive the susceptibility to fluoroquinolones.

Patients with severe dengue virus (DENV) infection can experience hemorrhage, shock and organ damage that coincides with defervescence and declining viremia. In addition to vascular leak, published work has shown that gut barrier permeability and elevated serum lipopolysaccharide (LPS) levels correlate with DENV disease severity. We previously described profound gut pathology in a mouse model of infection. This led to the hypothesis that a loss of gut epithelial barrier integrity and the influx of microbial products contribute to exacerbated inflammation and severe dengue disease. Consistent with this, here we demonstrated that DENV2 infection of AG129 mice compromised the gut epithelial barrier, promoted bacterial translocation, and induced microbial changes indicative of dysbiosis, including a reduced abundance of putatively beneficial microbial taxa and disrupted microbial circadian oscillations. Depleting gut bacteria with antibiotics from 1-day post-infection sharply reduced gut pathology as well as viral RNA in the proximal colon and liver, and the number of infected Kupffer cells in the liver. There was no evidence for systemic suppression of viral replication. We propose that viral infection of intestinal macrophages leads to inflammatory damage and mucosal barrier breakdown, dysbiosis and gut leak, with gut bacteria-dependent products promoting the susceptibility of Kupffer cells to DENV infection. Given the importance of liver damage in severe and fatal dengue disease, therapies that protect the gut barrier may help reduce disease severity.

Coxiella burnetii ( Cb ), the causative agent of Q fever, replicates within host macrophages by modulating innate immune responses through its type IVB secretion system (T4SS). Host cells sense pathogens via Pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), to recognize pathogen-associated molecular patterns (PAMPs) and initiate signaling pathways that drive pro-inflammatory cytokine and interferon responses. Toll-like receptor 3 (TLR3) triggers the nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-{kappa}B) and Interferon regulatory factors (IRFs) resulting in pro-inflammatory cytokine production and type I interferon (IFN-I) induction. Here we demonstrate that Cb requires T4SS to suppress TLR3-induced NF-{kappa}B and IRF transcriptional responses. Furthermore, RNA purified from both virulent and avirulent Cb is sufficient to activate TLR3, identifying pathogen RNA as a relevant PAMP in this context. Using a pulmonary infection model with virulent Cb, we found that TLR3/ Toll-Interleukin-1 Receptor Domain-Containing Adapter Protein Inducing Interferon Beta (TRIF)-dependent innate immune pathway inhibits cachexia. In contrast, signaling through the interferon-/{beta} receptor (IFNAR) restricts bacterial dissemination, indicating that these pathways play distinct but complementary roles in host defense. Mechanistically, Cb suppresses TLR3/TRIF signaling in a T4SS-dependent manner by preventing host TLR3 recruitment to the Coxiella-containing vacuole (CCV) and disrupting TRIF-TNF Receptor-Associated Factor 6 (TRAF6) interactions, thereby selectively inhibiting impairing NF-{kappa}B activation. Consistent with this, we identify five T4SS effector proteins that attenuate TLR3-induced NF-{kappa}B signaling, including CBU1292, which suppresses both NF-{kappa}B and IRF-dependent responses downstream of TLR3.

Bacterial infochemicals can profoundly alter phytoplankton physiology, yet how these compounds influence viral infection outcomes remains poorly understood. The bacterial quorum sensing molecule 2-heptyl-4-quinolone (HHQ) enables the globally abundant coccolithophore, Gephyrocapsa huxleyi, to evade virus-induced mortality following infection by Emiliania huxleyi virus (EhV). HHQ reduced infectious virus production at 24 hours despite largely intact viral gene transcription, as revealed by RNA sequencing of host and viral responses during EhV-201 infection. Host transcriptomics instead revealed rapid and progressive HHQ-dependent physiological remodeling evidenced by changes in protein encoding genes associated with vesicular trafficking, and cytoskeletal organization. Transcriptional responses further implicated endolysosomal sorting, membrane remodeling, and protein modification as potential barriers to productive infection. These findings indicate that HHQ-mediated evasion of virus-induced mortality occurs downstream of viral entry and transcription, through remodeling of host processes required for virion assembly, maturation, trafficking, and release.

Pseudohyphal growth in skin-associated fungi is often considered a morphological response to environmental stress, but whether it represents a coordinated metabolic state that influences host immunity remains unclear. Using Malassezia furfur, we combined synchronized morphogenesis, temporal metabolomics, stable-isotope tracing, targeted metabolite measurements, transcriptional profiling, and functional immune assays to compare yeast and pseudohyphal states. Yeast cells maintained an active tryptophan-aldehyde metabolic program that partitioned carbon between oxidative and reductive branches associated with redox balance and methylation potential. Pseudohyphal cells attenuated this program and instead underwent sequential metabolic remodeling, characterized by early changes in one-carbon and amino acid metabolism followed by sulfur-pathway remodeling and increased glutathione biosynthesis. These distinct fungal states generated extracellular environments that altered THP-1 innate immune responsiveness without directly inducing canonical macrophage polarization. Yeast-conditioned cells responded preferentially to tissue-repair signals, whereas pseudohypha-conditioned cells displayed enhanced responses to inflammatory stimuli. Individual metabolites did not reproduce these effects, suggesting that immune conditioning depends on the integrated extracellular milieu. Together, these findings identify pseudohyphal growth as an organized adaptive metabolic state and establish a framework linking fungal morphogenetic potential to context-dependent host immune responses.

Microsporidia are divergent fungal pathogens that infect a wide range of hosts. Microsporidia have evolved highly reduced genomes, resulting in the loss of many metabolic pathways. Encephalitozoon intestinalis is a human-infecting microsporidian species with the smallest known eukaryotic genome (2.3 Mbp). As an obligate intracellular parasite, E. intestinalis is dependent on its host for replication. Our current understanding of E. intestinalis cell biology remains limited. This is largely due to genetic intractability, which has made live-cell imaging challenging, and its small size (~2 m), which is within the diffraction limit of conventional microscopy. Here we develop a live-cell imaging technique, "silhouette microscopy", that does not rely on genetic manipulation of microsporidia, and enables us to study the live cell dynamics of E. intestinalis replication in host cells, from entry to exit. We report the timing of cell division, kinetics of the parasite life cycle, and dynamics of the parasitophorous vacuole within which E. intestinalis resides. The parasitophorous vacuole undergoes frequent fission and fusion, and ultimately ruptures to facilitate parasite release from the host cell. To gain a higher resolution understanding into the cell biology of E. intestinalis development, we performed ultrastructure expansion microscopy (U-ExM) on infected cells. U-ExM reveals the organization of the E. intestinalis cytoskeleton and development of the specialized invasion organelle, the polar tube. The combination of live-cell imaging and U-ExM provide a platform to study the E. intestinalis life cycle, and sheds light on the dynamics and cell biology of E. intestinalis in host cells.

Ultrasmall episymbiotic bacteria grow on the surface of their obligate bacterial hosts. These episymbionts have a restricted host range and likely rely on host-derived receptor molecules to identify and associate with cognate hosts. Nanosynbacteria from the oral microbiome, like Nanosynbacter lyticus, utilize a pair of distinct type 4 pili (T4P) for host surface interactions, T4P-1 and T4P-2. In this study we identify the adhesin components of both T4P, demonstrate that these adhesins localize to the cell wall of the cognate host Actinobacterium Schaalia odontolytica, and demonstrate that each adhesin binds distinct components of the host cell wall. The adhesin from the motility driving T4P-1, termed PilX1, showed a wide range of affinities in microarray screening and bound peptidoglycan directly, but could not bind purified cell wall polysaccharides (CWPS) from host surfaces. Conversely, the adhesin from T4P-2, termed PilX2, bound host-derived CWPS and preferentially bound episymbiont-naive hosts over episymbiont-exposed hosts. Within the bacterial host we identified two CWPS biosynthesis pathways that altered adhesin binding and episymbiont susceptibility. Disruption of a rhamnose-CWPS pathway left hosts susceptible to episymbiont binding and disruption of a fucose remodeling pathway made hosts more resistant to episymbiont binding. Analyzing polysaccharide compositions revealed multiple CWPS, potentially including a rhamnose- and mannose-rich rhamnomannan. Consistent with this finding, exogenously added rhamnose and mannose were able to block binding by N. lyticus, supporting a role for these sugars in episymbiont defense. Our study reveals that Nanosynbacteria rely on the complex sugars that coat their host bacteria for recognition and attachment.

The endonuclear symbiont Holospora obtusa invades the macronucleus of Paramecium caudatum using an infectious-form invasion-tip containing a major 89-kDa periplasmic protein implicated in escape from the host digestive vacuole and macronuclear invasion. Sequence-based analyses placed this protein within a Holospora-specific family and predicted an N-terminal membrane-anchoring module and a long alpha-helical C-terminal region. AlphaFold2 modeling followed by molecular dynamics indicated that the C-terminal region forms an antiparallel, spectrin-repeat-like helical solenoid containing a localized three-helix coiled-coil. The protein exhibits a large pH-dependent charge difference, becoming 55 units more positive at pH 4 than at pH 7. Independent simulations at these pH values differed mainly in the relative orientation of the N-terminal core and C-terminal rod, described by DynDom as an approximately 35 degrees rigid-body rotation about a hinge centered on Gly291. However, none of the nearby glutamates (Glu273, Glu275, and Glu302) formed a pH-dependent salt bridge, and no specific protonation-dependent interaction responsible for the conformational difference was identified. We nonetheless propose, as a testable hypothesis, that acidification of the digestive vacuole may promote this reorientation, thereby repositioning the N-terminal membrane-anchoring module at the bacterial surface as an early step toward escape from the vacuole.

During infection, Klebsiella pneumoniae must withstand host-derived oxidative stress not only by detoxifying reactive oxygen species (ROS) but also by repairing oxidative damage to cellular physiology. How these detoxification and stress-adaptive responses are coordinated remains unclear. Here, we identify a plasmid-encoded tacAT locus as an OxyR-responsive regulatory module that supports peroxide tolerance and in vivo fitness in K. pneumoniae. Guided by the conservation of the OxyR DNA-binding domain and its consensus binding motif, we surveyed promoter-proximal regions of the K. pneumoniae CRK3022 chromosome and plasmids for candidate OxyR-responsive loci. This analysis identified tacAT, a putative type II toxin-antitoxin (TA) module, as the only plasmid-associated candidate. OxyR directly bound the tacAT promoter and contributed to tacAT induction during hydrogen peroxide (H?O?) stress. The TacAT complex exhibited DNA-binding and transcriptional regulatory activity, promoting the expression of genes linked to protein quality control and membrane/envelope homeostasis, including clpB, htpG, cadC, bhsA, and marA. Deletion of tacAT compromised survival under lethal peroxide challenge and attenuated bacterial dissemination, tissue pathology, and inflammatory responses in a murine bacteremia model. These findings define an OxyR-TacAT regulatory branch that connects peroxide sensing with stress-adaptive gene expression, revealing how a TA-associated module can be integrated into stress-adaptive programs during host-associated oxidative stress.

Ralstonia pseudosolanacearum causes a lethal bacterial wilt disease in many plants. The pathogen enters through roots and systemically colonizes the xylem tissue, resulting in wilting and eventual death of the host. Using transposon mutagenesis followed by screening in tomato seedlings, we identified a mutant exhibiting virulence deficiency by root inoculation, but not by leaf inoculation. The mutation was recreated in the wild-type genome via natural transformation to prove the linkage of virulence deficiency with the transposon insertion. Whole genome sequencing revealed that the transposon insertion was in a cyp450 homologue, encoding cytochrome P450 monooxygenase. Two independent targeted mutations were generated in the cyp450 gene, and both the mutants recapitulated the root-specific virulence deficiency. The mutants rarely cause disease in mature plants following soil drenching but remained fully virulent upon petiole pricking inoculation. Complementation with the full length cyp450 gene amplified from the wild-type strain F1C1 restored virulence in the mutant. Compared to the wild-type F1C1, the cyp450 mutants exhibited greater sensitivity to tomato root extracts. HR-LCMS of tomato root extracts revealed the occurrence of several antimicrobial compounds. Cytochrome P450 enzymes are known to mediate the degradation or biosynthesis of diverse metabolites in bacteria. Considering the virulence difference due to the mode of inoculation, our findings indicate that cyp450 contributes to tissue-specific adaptation of the pathogen during infection has opened an avenue for integrating tissue metabolomics with bacterial wilt susceptibility research.

Peach shoot blight (PSB), caused by Diaporthe amygdali, is one of the most devastating fungal diseases affecting peach production worldwide. Glutaredoxins (Grxs) are ubiquitous redox-regulatory proteins involved in antioxidant defense and iron metabolism, yet their functions in phytopathogenic fungi remain largely unknown. Here, we identified four Grxs (DaGrxA-DaGrxD) in D. amygdali. Among them, DaGrxB, a monothiol glutaredoxin, is the sole member essential for vegetative growth, asexual reproduction and virulence. DaGrxB interacts with iron-regulatory transcription factors DaHapX and DaSreA and modulates their activities via iron-dependent nucleocytoplasmic shuttling, coordinating iron acquisition and storage gene expression. During infection, D. amygdali encounters a biphasic iron environment: an inherently iron-limited host environment at early stages (1-2 dpi) followed by host-mediated iron excess (3-4 dpi), and DaGrxB is essential for navigating this transition. Consistently, deletion of either DaHAPX or DaSREA significantly reduced virulence, confirming that iron homeostasis is a critical determinant of D. amygdali virulence. Notably, deletion of DaGRXB disrupts Fe-S cluster biosynthesis, causing mitochondrial Fe2+ accumulation, impaired aconitase activity, and reduced ATP production, which triggers constitutive activation of the GSH-dependent antioxidant system, with elevated GSH/GSSG ratios, increased GR and GST activities, and reduced H2O2 levels. Exogenous GSH and H2O2 depletion both reduced virulence, suggesting that disturbed redox balance directly contributes to attenuated virulence. Additionally, the Trx domain assists the Grx domain in nucleocytoplasmic translocation through intramolecular interactions. Finally, LDH-delivered dsRNA targeting DaGRXB effectively controls PSB. Collectively, these findings reveal how a monothiol glutaredoxin integrates iron homeostasis, redox balance and virulence in a plant pathogen, providing a foundation for RNAi-based disease management.

Serine protease inhibitors (serpins) are extremely rare in prokaryotes, and their role in immune evasion remains unclear. We identified a bifunctional TLR2-binding serpin (miropin) produced by Tannerella forsythia that broadly suppresses neutrophil antimicrobial capacity. Miropin binding to TLR2 primed neutrophils, enhancing phagocytic capacity yet, counterintuitively, promoting bacterial persistence. Mechanistically, like other serpins, miropin acted as an irreversible suicide inhibitor of neutrophil serine proteases (NSPs), blocking their activity upon granule mobilization and NETosis. Unexpectedly, miropin, independent of its protease-inhibitory activity, bound and inhibited myeloperoxidase (MPO), thereby blocking the oxidative burst. This simultaneous suppression of oxidative and non-oxidative bactericidal mechanisms promoted bacterial survival in vivo without significantly altering the tissue inflammatory response. Thus, we identified novel moonlighting roles (TLR2 signaling and MPO inhibition) for an unorthodox prokaryotic serpin and provided context for how T. forsythia and other oral pathogens survive within the subgingival niche, an environment highly enriched in activated neutrophils.

Mycobacterium tuberculosis has a flavin-intensive metabolism, with more than 180 proteins that require flavin or deazaflavin cofactors. M. tuberculosis requires riboflavin biosynthesis for growth in standard culture conditions in vitro, which can be overcome by providing exogenous riboflavin. It is unknown whether M. tuberculosis also requires riboflavin synthesis to grow or persist in infected host cells or tissues. Here we used a M. tuberculosis strain that conditionally expresses ribA2, which encodes a bifunctional enzyme that catalyzes the first steps in both branches of riboflavin biosynthesis, to determine whether M. tuberculosis requires riboflavin biosynthesis during infection. Transcriptional repression of ribA2 caused death of M. tuberculosis in vitro without riboflavin supplementation. RNAseq revealed that riboflavin starved M. tuberculosis induce a transcriptional program indicating decreased electron transport chain function and broad changes to central metabolism. Transcriptional repression of ribA2 prevented M. tuberculosis growth in cultured macrophages and caused clearance of M. tuberculosis from infected tissues at all stages of infection in both C57BL/6 mice that form cellular lung lesions and C3HeB/FeJ mice that develop necrotic lung granulomas. Our results demonstrate that M. tuberculosis requires RibA2 for viability at all stages of infection and support further characterization of the riboflavin biosynthesis pathway as a potential target for development of new anti-tubercular drugs.

Soil bacteria play a crucial role in supporting ecosystem health, yet little is known about how wild populations adapt to abiotic stressors, like limited water availability. We used 186 Luteibacter isolates from maize roots grown in never-irrigated prairie soils collected from a steep precipitation gradient in Kansas, US to test whether climate history is associated with osmotic stress tolerance and genomic differentiation. In vitro, strains from semi-arid sites were more tolerant to osmotic-stress treatment than those from humid sites. Comparative genomics of a subset of 96 isolates identified four strongly biogeographically structured lineages and extensive accessory-genome diversity. Semi-arid climate-associated strains were enriched for genes involved in osmotic homeostasis, oxidative stress protection, motility, and type IV secretion. Humid climate-associated strains were enriched in environmental sensing, metabolic versatility, biofilm formation, iron acquisition, type VI secretion, and resource competition. GWAS identified 22 candidate genes associated with osmotic-stress tolerance, including those related to type IV pili, biofilm production, DNA repair, and phage. No candidates were shared between semi-arid and humid strains, suggesting distinct genetic mechanisms of osmotic tolerance. Predicted prophage composition was also geographically structured, although total prophage gene content did not predict osmotic-stress tolerance. Together, these results show that adaptation to long-term differences in water availability likely involves coordinated shifts in stress physiology, genome content, and ecological interactions in wild bacterial populations, providing insight into predicting microbial responses to changing climates.

Spatial organization during bacterial fission is critical to ensure that division results in identical, viable progeny cells. In Escherichia coli, one important system that accomplishes this is the Min system, comprising MinC, MinD, and MinE proteins. The Min system regulates assembly of the FtsZ-ring to ensure proper placement of the division machinery at midcell. We explored the dynamics of the Min system and demonstrate that Min oscillation and release of MinD from the membrane is modulated by MinC. Deletion of the minC gene from a strain expressing minD fused to the gene encoding green fluorescent protein (Gfp) resulted in faster pole-to-pole oscillation of Gfp-MinD in vivo, leading to a shorter period. This was also observed in a strain expressing the MinC(R133A), a variant mutated at the MinC-MinD interface. In phospholipid binding and release assays in vitro, we observed that the concentration of MinE required to release MinD was lower with MinC, suggesting that MinC activates MinD for MinE-dependent release. Oscillation is produced by a diffusion-capture-release mechanism occurring at the membrane with proteins proceeding toward a pole. To determine if an increase in the diffusive proportion of MinD leads to faster oscillation, we constructed MinD membrane targeting sequence (MTS) variants to reduce the membrane associated pool of MinD. MTS variants induced a range of oscillation defects, with MinD(KR265AA) oscillating faster in vivo. Together our results show that MinC is an effector that modifies Min oscillation by priming MinD for MinE-dependent membrane release and fine-tunes the Min oscillation rate in dividing cells.

T follicular helper cells (Tfh) within germinal centers are among the most permissive cells for HIV infection and serve as a critical part of the viral reservoir. Given the key role of Tfh in affinity maturation and the production of plasma cells and memory B cells, an understanding of this critical viral reservoir is fundamental not only for cure strategies but also for understanding immunodeficiency in people living with HIV (PLWH). Using rare clinical specimens from PLWH, we demonstrate that HIV causes both a change in the cellular composition of germinal centers and changes in their overall architecture in ways that are consistent with the immunodeficiency seen in PLWH, independent of their CD4 count. We note a significant decrease in the number of GC Tfh cells and an increase in the number of follicular CD8 T cells in lymphoid tissues obtained from PLWH. Our data further show that GC size is decreased both by HIV and with increased age. Single-cell RNA sequencing data demonstrate that HIV acutely decreases the expression of Tfh B-cell support pathways and disrupts Tfh expression of cell trafficking receptors. Additionally, in a lymphoid organoid model of acute infection that forms GC-like structures, these findings are recapitulated, suggesting that New Approach Methodologies could be germane to understanding the impact of HIV on the reservoir and immunodeficiency. This study describes previously undocumented remodeling of the germinal center niche in PLWH, providing insight into the immune microenvironment of a key HIV reservoir that may help inform future cure strategies.

Integrative and conjugative elements (ICEs) are widespread in bacteria and typically confer important phenotypes to their hosts. Following excision from the host chromosome, an ICE can transfer to a new cell through the element-encoded conjugation machinery (a type IV secretion system) to create a transconjugant. Tn916, a well-studied ICE that confers tetracycline resistance, is found in several Gram-positive pathogens and has been widely studied in Bacillus subtilis. Unlike most ICEs, Tn916 causes host-cell death following its activation (excision and gene expression). We found that orf7 and orf8 (renamed sig7 and sig8), two regulatory genes in Tn916, encode a two-subunit (split) sigma factor of RNA polymerase, Sig7-8, that activates transcription from Porf7 (now Psig7) and is required for excision of the element. Importantly, production of Sig7-8, either from Psig7 or another strong promoter, caused cell death. Using a Cre recombinase reporter to detect gene expression, we detected little or no transcription from Psig7 in transconjugants. This lack of transcription from Psig7 in transconjugants was essential for fitness of both the element and its host: forcing transcription from Psig7 in transconjugants caused either loss of Tn916 or death of nascent transconjugants. Our findings indicate that, unlike other well-studied ICEs (e.g., ICEBs1) that use repressors to control gene expression and are expressed in nascent transconjugants, most Tn916 genes remain off following transfer to a new host. This regulation prevents Tn916 from killing transconjugants and allows successful horizontal transfer and maintains the potential for future activation in the new host.