Type VI secretion systems promote intraspecific competition and host interactions in a bee gut symbiont.

The Type VI Secretion System (T6SS) is a sophisticated mechanism utilized by gram-negative bacteria to deliver toxic effector proteins into target cells, influencing microbial community dynamics and host interactions. In this study, we investigated the role of T6SSs in Snodgrassella alvi wkB2, a core bacterial symbiont of the honey bee gut microbiota. We generated single- and double-knockout mutants targeting essential genes (tssD and tssE) in both T6SS-1 and T6SS-2 and assessed their colonization and competition capabilities in vivo. Our results indicate that T6SSs are nonessential for colonization of the bee gut, although T6SS-2 mutant strains exhibited significantly lower colonization levels compared to the wild-type (WT) strain. Further, a defined community experiment showed that S. alvi wkB2 T6SSs do not significantly impact interspecific competition among core gut bacteria. However, cocolonization experiments with closely related S. alvi strains demonstrated that T6SS-1 plays a role in mediating intraspecific competition. Transcriptomic analysis of bee guts monocolonized by WT or T6SS mutants revealed differential expression of host immunity-related genes relative to microbiota-deprived bees, such as upregulation of the antimicrobial peptide apidaecin in the presence of WT S. alvi and the antimicrobial peptide defensin in the presence of T6SS-2 mutant S. alvi, suggesting that T6SSs contribute to shaping host immune responses. These findings provide insight into the ecological roles of T6SSs in the honey bee gut microbiota, emphasizing their importance in maintaining competitive dynamics and influencing host-bacterial interactions.

Engineered gut symbiont inhibits microsporidian parasite and improves honey bee survival.

Honey bees (Apis mellifera) are critical agricultural pollinators as well as model organisms for research on development, behavior, memory, and learning. The parasite Nosema ceranae, a common cause of honey bee colony collapse, has developed resistance to small-molecule therapeutics. An alternative long-term strategy to combat Nosema infection is therefore urgently needed, with synthetic biology offering a potential solution. Honey bees harbor specialized bacterial gut symbionts that are transmitted within hives. Previously, these have been engineered to inhibit ectoparasitic mites by expressing double-stranded RNA (dsRNA) targeting essential mite genes, via activation of the mite RNA interference (RNAi) pathway. In this study, we engineered a honey bee gut symbiont to express dsRNA targeting essential genes of N. ceranae via the parasite's own RNAi machinery. The engineered symbiont sharply reduced Nosema proliferation and improved bee survival following the parasite challenge. This protection was observed in both newly emerged and older forager bees. Furthermore, engineered symbionts were transmitted among cohoused bees, suggesting that introducing engineered symbionts to hives could result in colony-level protection.

Agrobacterium tumefaciens divisome proteins regulate the transition from polar growth to cell division.

The mechanisms that restrict peptidoglycan biosynthesis to the pole during elongation and re-direct peptidoglycan biosynthesis to mid-cell during cell division in polar-growing Alphaproteobacteria are largely unknown. Here, we explore the role of early division proteins of Agrobacterium tumefaciens including three FtsZ homologs, FtsA and FtsW in the transition from polar growth to mid-cell growth and ultimately cell division. Although two of the three FtsZ homologs localize to mid-cell, exhibit GTPase activity and form co-polymers, only one, FtsZAT , is required for cell division. We find that FtsZAT is required not only for constriction and cell separation, but also for initiation of peptidoglycan synthesis at mid-cell and cessation of polar peptidoglycan biosynthesis. Depletion of FtsZAT in A. tumefaciens causes a striking phenotype: cells are extensively branched and accumulate growth active poles through tip splitting events. When cell division is blocked at a later stage by depletion of FtsA or FtsW, polar growth is terminated and ectopic growth poles emerge from mid-cell. Overall, this work suggests that A. tumefaciens FtsZ makes distinct contributions to the regulation of polar growth and cell division.

FzlA, an essential regulator of FtsZ filament curvature, controls constriction rate during Caulobacter division.

During bacterial division, polymers of the tubulin-like GTPase FtsZ assemble at midcell to form the cytokinetic Z-ring, which coordinates peptidoglycan (PG) remodeling and envelope constriction. Curvature of FtsZ filaments promotes membrane deformation in vitro, but its role in division in vivo remains undefined. Inside cells, FtsZ directs PG insertion at the division plane, though it is unclear how FtsZ structure and dynamics are mechanistically coupled to PG metabolism. Here we study FzlA, a division protein that stabilizes highly curved FtsZ filaments, as a tool for assessing the contribution of FtsZ filament curvature to constriction. We show that in Caulobacter crescentus, FzlA must bind to FtsZ for division to occur and that FzlA-mediated FtsZ curvature is correlated with efficient division. We observed that FzlA influences constriction rate, and that this activity is associated with its ability to bind and curve FtsZ polymers. Further, we found that a slowly constricting fzlA mutant strain develops 'pointy' poles, suggesting that FzlA influences the relative contributions of radial versus longitudinal PG insertion at the septum. These findings implicate FzlA as a critical coordinator of envelope constriction through its interaction with FtsZ and suggest a functional link between FtsZ curvature and efficient constriction in C. crescentus.

One-step genome engineering in bee gut bacterial symbionts.

Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiont Snodgrassella alvi. We are also able to engineer the genomes of multiple strains of S. alvi and another species, Snodgrassella communis, which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont, Bartonella apis, which is an alphaproteobacterium. As expected, gene knockout in S. alvi using this approach is recA-dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods. IMPORTANCE: Honey bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health.

Virulence-linked adhesin drives mutualist colonization of the bee gut via biofilm formation.

Bacterial biofilms are stable multicellular structures that can enable long term host association. Yet, the role of biofilms in supporting gut mutualism is still not fully understood. Here, we investigate Snodgrassella alvi , a beneficial bacterial symbiont of honey bees, and find that biofilm formation is required for its colonization of the bee gut. We constructed fifteen S. alvi mutants containing knockouts of genes known to promote colonization with putative roles in biofilm formation. Genes required for colonization included staA and staB , encoding trimeric autotransporter adhesins (TAAs) and mltA , encoding a lytic transglycosylase. Intriguingly, TAAs are considered virulence factors in pathogens but support mutualism by the symbiont S. alvi. In vitro , biofilm formation was reduced in Δ staB cells and abolished in the other two mutants. Loss of staA also reduced auto-aggregation and cell-cell connections. Based on structural predictions, StaA/B are massive (>300 nm) TAAs with many repeats in their stalk regions. Further, we find that StaA/B are conserved across Snodgrassella species, suggesting that StaA/B-dependent colonization is characteristic of this symbiont lineage. Finally, staA deletion increases sensitivity to bactericidal antimicrobials, suggesting that the biofilm indirectly buffers against antibiotic stress. In all, the inability of two biofilm-deficient strains (Δ staA and Δ mltA ) to effectively mono-colonize bees indicates that S. alvi biofilm formation is required for colonization of the bee gut. We envision the bee gut system as a genetically tractable model for studying the physical basis of biofilm-mutualist-gut interactions.

Honey bee functional genomics using symbiont-mediated RNAi.

Honey bees are indispensable pollinators and model organisms for studying social behavior, development and cognition. However, their eusociality makes it difficult to use standard forward genetic approaches to study gene function. Most functional genomics studies in bees currently utilize double-stranded RNA (dsRNA) injection or feeding to induce RNAi-mediated knockdown of a gene of interest. However, dsRNA injection is laborious and harmful, and dsRNA feeding is difficult to scale cheaply. Further, both methods require repeated dsRNA administration to ensure a continued RNAi response. To fill this gap, we engineered the bee gut bacterium Snodgrassella alvi to induce a sustained host RNA interference response that reduces expression of a targeted gene. To employ this functional genomics using engineered symbionts (FUGUES) procedure, a dsRNA expression plasmid is cloned in Escherichia coli using Golden Gate assembly and then transferred to S. alvi. Adult worker bees are then colonized with engineered S. alvi. Finally, gene knockdown is verified through qRT-PCR, and bee phenotypes of interest can be further assessed. Expression of targeted genes is reduced by as much as 50-75% throughout the entire bee body by 5 d after colonization. This protocol can be accomplished in 4 weeks by bee researchers with microbiology and molecular cloning skills. FUGUES currently offers a streamlined and scalable approach for studying the biology of honey bees. Engineering other microbial symbionts to influence their hosts in ways that are similar to those described in this protocol may prove useful for studying additional insect and animal species in the future.

Single-step genome engineering in the bee gut symbiont Snodgrassella alvi.

Honey bees are economically relevant pollinators experiencing population declines due to a number of threats. As in humans, the health of bees is influenced by their microbiome. The bacterium Snodgrassella alvi is a key member of the bee gut microbiome and has a role in excluding pathogens. Despite this importance, there are not currently any easy-to-use methods for modifying the S. alvi chromosome to study its genetics. To solve this problem, we developed a one-step procedure that uses electroporation and homologous recombination, which we term SnODIFY (Snodgrassella-specific One-step gene Deletion or Insertion to alter FunctionalitY). We used SnODIFY to create seven single-gene knockout mutants and recovered mutants for all constructs tested. Nearly all transformants had the designed genome modifications, indicating that SnODIFY is highly accurate. Mutant phenotypes were validated through knockout of Type 4 pilus genes, which led to reduced biofilm formation. We also used SnODIFY to insert heterologous sequences into the genome by integrating fluorescent protein-coding genes. Finally, we confirmed that genome modification is dependent on S. alvi's endogenous RecA protein. Because it does not require expression of exogenous recombination machinery, SnODIFY is a straightforward, accurate, and lightweight method for genome editing in S. alvi. This workflow can be used to study the functions of S. alvi genes and to engineer this symbiont for applications including protection of honey bee health.

Role of sphingolipids in murine radiation-induced lung injury: protection by sphingosine 1-phosphate analogs.

Clinically significant radiation-induced lung injury (RILI) is a common toxicity in patients administered thoracic radiotherapy. Although the molecular etiology is poorly understood, we previously characterized a murine model of RILI in which alterations in lung barrier integrity surfaced as a potentially important pathobiological event and genome-wide lung gene mRNA levels identified dysregulation of sphingolipid metabolic pathway genes. We hypothesized that sphingolipid signaling components serve as modulators and novel therapeutic targets of RILI. Sphingolipid involvement in murine RILI was confirmed by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to sphingosine 1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage fluid, and lung tissue. Mice with a targeted deletion of SphK1 (SphK1(-/-)) or with reduced expression of S1P receptors (S1PR1(+/-), S1PR2(-/-), and S1PR3(-/-)) exhibited marked RILI susceptibility. Finally, studies of 3 potent vascular barrier-protective S1P analogs, FTY720, (S)-FTY720-phosphonate (fTyS), and SEW-2871, identified significant RILI attenuation and radiation-induced gene dysregulation by the phosphonate analog, fTyS (0.1 and 1 mg/kg i.p., 2×/wk) and to a lesser degree by SEW-2871 (1 mg/kg i.p., 2×/wk), compared with those in controls. These results support the targeting of S1P signaling as a novel therapeutic strategy in RILI.

Computational identification of a systemic antibiotic for gram-negative bacteria.

Discovery of antibiotics acting against Gram-negative species is uniquely challenging due to their restrictive penetration barrier. BamA, which inserts proteins into the outer membrane, is an attractive target due to its surface location. Darobactins produced by Photorhabdus, a nematode gut microbiome symbiont, target BamA. We reasoned that a computational search for genes only distantly related to the darobactin operon may lead to novel compounds. Following this clue, we identified dynobactin A, a novel peptide antibiotic from Photorhabdus australis containing two unlinked rings. Dynobactin is structurally unrelated to darobactins, but also targets BamA. Based on a BamA-dynobactin co-crystal structure and a BAM-complex-dynobactin cryo-EM structure, we show that dynobactin binds to the BamA lateral gate, uniquely protruding into its ß-barrel lumen. Dynobactin showed efficacy in a mouse systemic Escherichia coli infection. This study demonstrates the utility of computational approaches to antibiotic discovery and suggests that dynobactin is a promising lead for drug development.

Simvastatin attenuates radiation-induced murine lung injury and dysregulated lung gene expression.

Novel therapies are desperately needed for radiation-induced lung injury (RILI), which, despite aggressive corticosteroid therapy, remains a potentially fatal and dose-limiting complication of thoracic radiotherapy. We assessed the utility of simvastatin, an anti-inflammatory and lung barrier-protective agent, in a dose- and time-dependent murine model of RILI (18-(25 Gy). Simvastatin reduced multiple RILI indices, including vascular leak, leukocyte infiltration, and histological evidence of oxidative stress, while reversing RILI-associated dysregulated gene expression, including p53, nuclear factor-erythroid-2-related factor, and sphingolipid metabolic pathway genes. To identify key regulators of simvastatin-mediated RILI protection, we integrated whole-lung gene expression data obtained from radiated and simvastatin-treated mice with protein-protein interaction network analysis (single-network analysis of proteins). Topological analysis of the gene product interaction network identified eight top-prioritized genes (Ccna2a, Cdc2, fcer1 g, Syk, Vav3, Mmp9, Itgam, Cd44) as regulatory nodes within an activated RILI network. These studies identify the involvement of specific genes and gene networks in RILI pathobiology, and confirm that statins represent a novel strategy to limit RILI.

The novel role of the mu opioid receptor in lung cancer progression: a laboratory investigation.

BACKGROUND: The possibility that µ opioid agonists can influence cancer recurrence is a subject of recent interest. Epidemiologic studies suggested that there were differences in cancer recurrence in breast and prostate cancer contingent on anesthetic regimens. In this study, we identify a possible mechanism for these epidemiologic findings on the basis of µ opioid receptor (MOR) regulation of Lewis lung carcinoma (LLC) tumorigenicity in cell and animal models. METHODS: We used human lung tissue and human non-small cell lung cancer (NSCLC) cell lines and evaluated MOR expression using immunoblot and immunohistochemical analysis. LLC cells were treated with the peripheral opioid antagonist methylnaltrexone (MNTX) or MOR shRNA and evaluated for proliferation, invasion, and soft agar colony formation in vitro and primary tumor growth and lung metastasis in C57BL/6 and MOR knockout mice using VisEn fluorescence mediated tomography imaging and immunohistochemical analysis. RESULTS: We provide several lines of evidence that the MOR may be a potential target for lung cancer, a disease with high mortality and few treatment options. We first observed that there is ∼5- to 10-fold increase in MOR expression in lung samples from patients with NSCLC and in several human NSCLC cell lines. The MOR agonists morphine and [D-Ala(2), N-MePhe(4), Gly-ol]-enkephalin (DAMGO) increased in vitro LLC cell growth. Treatment with MNTX or silencing MOR expression inhibited LLC invasion and anchorage-independent growth by 50%-80%. Injection of MOR silenced LLC lead to a ∼65% reduction in mouse lung metastasis. In addition, MOR knockout mice do not develop significant tumors when injected with LLC in comparison with wild-type controls. Finally, continuous infusion of the peripheral opioid antagonist MNTX attenuates primary LLC tumor growth and reduces lung metastasis. CONCLUSIONS: Taken together, our data suggest a possible direct effect of opiates on lung cancer progression, and provide a plausible explanation for the epidemiologic findings. Our observations further suggest a possible therapeutic role for opioid antagonists.

An Essential Regulator of Bacterial Division Links FtsZ to Cell Wall Synthase Activation.

Bacterial growth and division require insertion of new peptidoglycan (PG) into the existing cell wall by PG synthase enzymes. Emerging evidence suggests that many PG synthases require activation to function; however, it is unclear how activation of division-specific PG synthases occurs. The FtsZ cytoskeleton has been implicated as a regulator of PG synthesis during division, but the mechanisms through which it acts are unknown. Here, we show that FzlA, an FtsZ-binding protein and essential regulator of constriction in Caulobacter crescentus, helps link FtsZ to PG synthesis to promote division. We find that hyperactive mutants of the PG synthases FtsW and FtsI specifically render fzlA, but not other division genes, non-essential. However, FzlA is still required to maintain proper constriction rate and efficiency in a hyperactive PG synthase background. Intriguingly, loss of fzlA in the presence of hyperactivated FtsWI causes cells to rotate about the division plane during constriction and sensitizes cells to cell-wall-specific antibiotics. We demonstrate that FzlA-dependent signaling to division-specific PG synthesis is conserved in another α-proteobacterium, Agrobacterium tumefaciens. These data establish that FzlA helps link FtsZ to cell wall remodeling and is required for signaling to both activate and spatially orient PG synthesis during division. Overall, our findings support the paradigm that activation of SEDS-PBP PG synthases is a broadly conserved requirement for bacterial morphogenesis.

Growth factor release from a chemically modified elastomeric poly(1,8-octanediol-co-citrate) thin film promotes angiogenesis in vivo.

The ultimate success of in vivo organ formation utilizing ex vivo expanded "starter" tissues relies heavily upon the level of vascularization provided by either endogenous or artificial induction of angiogenic or vasculogenic events. To facilitate proangiogenic outcomes and promote tissue growth, an elastomeric scaffold previously shown to be instrumental in the urinary bladder regenerative process was modified to release proangiogenic growth factors. Carboxylic acid groups on poly(1,8-octanediol-co-citrate) films (POCfs) were modified with heparan sulfate creating a heparan binding POCf (HBPOCf). Release of proangiogenic growth factors vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and insulin-like growth factor 1 (IGF-1) from HBPOCfs demonstrated an approximate threefold increase over controls during a 30-day time course in vitro. Atomic force microscopy demonstrated significant topological differences between films. Subcutaneous implantation of POCf alone, HBPOCf, POCf-VEGF, and HBPOCf-VEGF within the dorsa of nude rats yielded increased vascular growth in HBPOCf-VEGF constructs. Vessel quantification studies revealed that POCfs alone contained 41.1 ± 4.1 vessels/mm², while HBPOCf, POCf-VEGF, and HBPOCF-VEGF contained 41.7 ± 2.6, 76.3 ± 9.4, and 167.72 ± 15.3 vessels/mm², respectively. Presence of increased vessel growth was demonstrated by CD31 and vWF immunostaining in HBPOCf-VEGF implanted areas. Data demonstrate that elastomeric POCfs can be chemically modified and possess the ability to promote angiogenesis in vivo.