Biology subjects

Mougous, J. D.

Publications and source records attributed to Mougous, J. D..

15 recordsLinked to original sources

bioRxiv2026

Host Cell Wall Polysaccharides Modulate Nanosynbacteria Recognition and Attachment

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.

microbiology↗

bioRxiv2026

Oral Actinobacteria sense and defend against parasitic epibionts

Bacteria contend with a wide range of antagonistic interactions from neighboring microbes. One widespread but poorly understood threat is posed by Patescibacteria, which proliferate by colonizing host bacteria surfaces and extracting cellular resources. Despite the ubiquity of Patescibacteria in the environment, how hosts defend against these epibionts remains unknown. Here we show that Actinomycetota fend off Patescibacteria using the Esx secretion system. We identify a secreted effector protein that mediates this defensive behavior and demonstrate that Esx induction is part of a multifaceted, epibiont-specific response that includes multiple predicted cell surface modifications. Finally, we show that this response occurs at the transcriptional level and is mediated by a previously undescribed threat sensing pathway whose activation leads to threonine phosphorylation of an FHA domain-containing output protein. Together with bioinformatic evidence linking this threat sensing pathway to Esx across Actinomycetales, our experimental findings establish a functional role for the elusive and broadly distributed Esx pathway in defense against epibionts. Furthermore, they help resolve the ecological relationship between Patescibacteria and their hosts as one of antagonism, not mutualism.

microbiology↗

bioRxiv2026

Orally available designed miniproteins inhibit enterotoxigenic Bacteroides fragilis pathology by blocking toxin receptor binding

Toxigenic bacterial infections in the gut are a significant contributor to the global burden of disease. Advanced tools for protein and live biotherapeutic engineering offer potentially transformative strategies for treating such diseases, while avoiding the collateral effects of traditional antibacterials. Here we used de novo protein design to identify inhibitors of the metzincin family protease Bacteroides fragilis toxin (BFT). These inhibitors, which bind distal to the active site, interfere with toxin-mediated E-cadherin cleavage and downstream proinflammatory signaling by blocking claudin-4 receptor binding. We tested the inhibitors as disulfide-stabilized variants administered directly to the cecum or in drinking water, as well as through in situ secretion by an engineered live biotherapeutic. Across these delivery modalities, the inhibitors successfully neutralized the toxin and effectively prevented BFT-associated gut pathology, including tumor formation. These results highlight the potential of de novo designed proteins as precise, non-antibiotic interventions to mitigate bacterial toxin-driven disease in the gut.

microbiology↗

bioRxiv2025

The type VI secretion system governs strain maintenance in a wild mammalian gut microbiome

Bacteria inhabiting the mammalian gut coexist in dense communities where contact-dependent antagonism mechanisms are widespread. The type VI secretion system (T6SS) is an interbacterial toxin delivery pathway prevalent among gut Bacteroidales, yet its function in naturally evolved microbiomes remains poorly defined. Here, we examine the role of the T6SS in Bacteroides within a physiologically relevant gut community derived from wild mice (the WildR microbiome). Using newly developed genetic tools and a strategy for functional replacement of strains within the WildR community, we demonstrate that the WildR isolate B. acidifaciens employs a T6SS to antagonize co-resident Bacteroidales. We also show that loss of T6SS function compromises the long-term maintenance of B. acidifaciens in the community but not its initial colonization, establishing the system as a determinant of strain persistence. The T6SS we identified resides on an integrative and conjugative element (ICE). ICE-seq, a targeted sequencing approach, reveals that the T6SS-ICE is distributed among select Bacteroidales and Muribaculaceae species in the WildR microbiome, between which it appears to be recently exchanged. We also show that transfer of the T6SS-ICE to WildR isolate Phocaeicola vulgatus confers transient colonization benefits in mice, but is linked to eventual population decline. Our findings demonstrate that the T6SS can stabilize the presence of specific strains within a complex, co-evolved gut microbiome, yet its value is context dependent and constrained by the ecological and physiological landscape of the host community.

microbiology↗

bioRxiv2025

De novo design of high-affinity miniprotein binders targeting Francisella tularensis virulence factor

Francisella tularensis poses considerable public health risk due to its high infectivity and potential for bioterrorism. Francisella-like lipoprotein (Flpp3), a key virulence factor unique to Francisella, plays critical roles in infection and immune evasion, making it a promising target for therapeutic development. However, the lack of well-defined binding pockets and structural information on native interactions has hindered structure-guided ligand discovery against Flpp3. Here, we used a combination of physics-based and deep-learning methods to design high-affinity miniprotein binders targeting two distinct sites on Flpp3. We identified four binders for site I with binding affinities ranging between 24-110 nM. For the second site, an initial binder showed a dissociation constant (KD) of 81 nM, and subsequent site saturation mutagenesis yielded variants with sub-nanomolar affinities. Circular dichroism confirmed the topology of designed miniproteins. The X-ray crystal structure of Flpp3 in complex with a site I binder is nearly identical to the design model (C root-mean-square deviation: 0.9 [A]). These designed miniproteins provide research tools to explore the roles of Flpp3 in tularemia and should enable the development of new therapeutic candidates.

biochemistry↗

bioRxiv2025

Recognition of unique cell surface glycopolymers by diversified lectin domains specifies umbrella toxin targeting

The bacterial cell envelope provides structural integrity and is an essential conduit through which the organism interacts with its environment. However, the molecular structures of its constituents can also be exploited as receptors, permitting threats such as toxins and phage access to the cell. We previously demonstrated that Streptomyces coelicolor secretes an umbrella toxin particle that acts in a highly selective manner to inhibit the hyphal growth of competing Streptomyces strains. Here, we identify the receptor of umbrella toxins as teichuronic acid (TUA) oligosaccharides anchored to the cell surface through linkage to wall teichoic acids (WTA). We show that the carbohydrate portion of this previously undescribed hybrid TUA-WTA molecule is variable across species and that targeting specificity derives from its selective recognition by diversified lectin domains associated with umbrella particles. A cryo-EM structure of a lectin-TUA complex reveals the molecular basis for umbrella toxin cell targeting and, in conjunction with bioinformatic analyses, provides insights into a molecular arms race that we posit drives diversification of umbrella particles and the chemical composition of Streptomyces cell envelopes.

microbiology↗

bioRxiv2025

Pseudomonads coordinate innate defense against viruses and bacteria with a single regulatory system

Bacterial cells live under the constant existential threats imposed by other bacteria and viruses. Their mechanisms for contending with these threats are well documented; however, the regulation of these diverse defense elements remains poorly understood. Here we show that bacteria can mount a genome-wide, coordinated, and highly effective immune response against bacterial and viral threats using a single regulatory pathway. Bioinformatic analyses revealed that Pseudomonas species broadly possess a specialized form of the Gac/Rsm regulatory pathway (GRP), which our prior work in Pseudomonas aeruginosa implicated in activating interbacterial antagonism defense mechanisms in response to neighbor cell death. Proteomic studies comparing GRP-activated and -inactivated strains derived from diverse Pseudomonas species showed that the pathway regulates a large and variable suite of factors implicated in defense against both bacterial and phage threats. Focusing on P. protegens, we identify profound phenotypic consequences of these factors against multiple forms of bacterial antagonism and several phage. Together, our results reveal that bacteria, like more complex organisms, couple danger sensing to the activation of an immune system with antibacterial and antiviral arms.

microbiology↗

bioRxiv2024

Essential and virulence-related protein interactions of pathogens revealed through deep learning

Identification of bacterial protein-protein interactions and predicting the structures of the complexes could aid in the understanding of pathogenicity mechanisms and developing treatments for infectious diseases. Here, we developed a deep learning-based pipeline that leverages residue-residue coevolution and protein structure prediction to systematically identify and structurally characterize protein-protein interactions at the proteome-wide scale. Using this pipeline, we searched through 78 million pairs of proteins across 19 human bacterial pathogens and identified 1923 confidently predicted complexes involving essential genes and 256 involving virulence factors. Many of these complexes were not previously known; we experimentally tested 12 such predictions, and half of them were validated. The predicted interactions span core metabolic and virulence pathways ranging from post-transcriptional modification to acid neutralization to outer membrane machinery and should contribute to our understanding of the biology of these important pathogens and the design of drugs to combat them.

biochemistry↗

bioRxiv2023

Umbrella toxin particles produced by Streptomyces block mycelial growth of competing species

The Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species. Here we show that in addition to small molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-EM structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, leading to our naming them umbrella particles. S. coelicolor encodes three umbrella particles with distinct toxin and lectin composition, and supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, S. griseus, we find inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative mycelial growth. Our data show that Streptomyces umbrella particles mediate competition between vegetative mycelia of related species, a function distinct from small molecule antibiotics, which are produced at the onset of reproductive growth and act broadly. Sequence analyses suggest this role of umbrella particles extends beyond Streptomyces, as we find umbrella loci in nearly one-thousand species across Actinobacteria.

microbiology↗

bioRxiv2023

Genetic manipulation of candidate phyla radiation bacteria provides functional insights into microbial dark matter

The study of bacteria has yielded fundamental insights into cellular biology and physiology, biotechnological advances and many therapeutics. Yet due to a lack of suitable tools, the significant portion of bacterial diversity held within the candidate phyla radiation (CPR) remains inaccessible to such pursuits. Here we show that CPR bacteria belonging to the phylum Saccharibacteria exhibit natural competence. We exploit this property to develop methods for their genetic manipulation, including the insertion of heterologous sequences and the construction of targeted gene deletions. Imaging of fluorescent protein-labeled Saccharibacteria provides high spatiotemporal resolution of phenomena accompanying epibiotic growth and a transposon insertion sequencing genome-wide screen reveals the contribution of enigmatic Saccharibacterial genes to growth on their Actinobacteria hosts. Finally, we leverage metagenomic data to provide cutting-edge protein structure-based bioinformatic resources that support the strain Southlakia epibionticum and its corresponding host, Actinomyces israelii, as a model system for unlocking the molecular underpinnings of the epibiotic lifestyle.

microbiology↗

bioRxiv2023

Discovery of a unique pathway for glutathione utilization in Francisella

Glutathione (GSH) is an abundant metabolite that can act as a signal, a nutrient source, or serve in a redox capacity for intracellular pathogens. For Francisella, GSH is thought to be a critical in vivo source of cysteine; however, the cellular pathways permitting GSH utilization by Francisella differ between strains and have remained poorly understood. Using genetic screening, we discovered a unique pathway for GSH utilization in Francisella spp. Whereas prior work suggested GSH catabolism initiates in the periplasm, the pathway we define consists of a major facilitator superfamily member that transports intact GSH and a previously unrecognized bacterial cytoplasmic enzyme that catalyzes the first step of GSH degradation. Interestingly, we find that the transporter gene for this pathway is pseudogenized in pathogenic Francisella, explaining phenotypic discrepancies in GSH utilization among Francisella spp. and revealing a critical role for GSH in the environmental niche of these bacteria.

microbiology↗

bioRxiv2021

Omnipose: a high-precision morphology-independent solution for bacterial cell segmentation

Advances in microscopy hold great promise for allowing quantitative and precise readouts of morphological and molecular phenomena at the single cell level in bacteria. However, the potential of this approach is ultimately limited by the availability of methods to perform unbiased cell segmentation, defined as the ability to faithfully identify cells independent of their morphology or optical characteristics. In this study, we present a new algorithm, Omnipose, which accurately segments samples that present significant challenges to current algorithms, including mixed bacterial cultures, antibiotic-treated cells, and cells of extended or branched morphology. We show that Omnipose achieves generality and performance beyond leading algorithms and its predecessor, Cellpose, by virtue of unique neural network outputs such as the gradient of the distance field. Finally, we demonstrate the utility of Omnipose in the characterization of extreme morphological phenotypes that arise during interbacterial antagonism and on the segmentation of non-bacterial objects. Our results distinguish Omnipose as a uniquely powerful tool for answering diverse questions in bacterial cell biology.

microbiology↗

bioRxiv2021

Coordinately regulated interbacterial antagonism defense pathways constitute a bacterial innate immune system

Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells1-3 4. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously we showed that Pseudomonas aeruginosa possess a danger sensing pathway that is a critical fitness determinant during competition against other bacteria5, 6. Here, we conducted genome-wide screens in P. aeruginosa that reveal three conserved and widespread interbacterial antagonism resistance clusters (arc1-3). We find that although arc1-3 are coordinately activated by the Gac/Rsm danger sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of P. aeruginosa to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.

microbiology↗

bioRxiv2020

An interbacterial DNA deaminase toxin directly mutagenizes surviving target populations

When bacterial cells come in contact, antagonism mediated by the delivery of toxins frequently ensues. The potential for such encounters to have long-term beneficial consequences in recipient cells has not been investigated. Here we examined the effects of intoxication by DddA, a cytosine deaminase delivered via the type VI secretion system (T6SS) of Burkholderia cenocepacia. Despite its killing potential, we observed that several bacterial species resist DddA and instead accumulate mutations installed by the toxin, indicating that even in the absence of killing, interbacterial toxins can have profound consequences on target populations. Investigation of additional toxins from the deaminase superfamily revealed that mutagenic activity is a common feature of these proteins, including a representative we show targets single-stranded DNA and displays a markedly divergent structure. Our findings suggest that a surprising consequence of antagonistic interactions between bacteria could be the promotion of adaptation via the action of directly mutagenic toxins.

microbiology↗

bioRxiv2017

Ixodes scapularis does not harbor a stable midgut microbiome

Hard ticks of the order Ixodidae serve as vectors for numerous human pathogens, including the causative agent of Lyme Disease Borrelia burgdorferi. Tick-associated microbes can influence pathogen colonization, offering the potential to inhibit disease transmission through engineering of the tick microbiota. Here, we investigate whether B. burgdorferi encounters abundant bacteria within the midgut of wild adult Ixodes scapularis, its primary vector. Through the use of controlled sequencing methods and confocal microscopy, we find that the majority of field-collected adult I. scapularis harbor limited internal microbial communities that are dominated by endosymbionts. A minority of I. scapularis ticks harbor abundant midgut bacteria and lack B. burgdorferi. We find that the lack of a stable resident midgut microbiota is not restricted to I. scapularis since extension of our studies to I. pacificus, Amblyomma maculatum, and Dermacentor spp showed similar patterns. Finally, bioinformatic examination of the B. burgdorferi genome revealed the absence of genes encoding known interbacterial interaction pathways, a feature unique to the Borrelia genus within the phylum Spirochaetes. Our results suggest that reduced selective pressure from limited microbial populations within ticks may have facilitated the evolutionary loss of genes encoding interbacterial competition pathways from Borrelia.

microbiology↗