Biology subjects

Grossman, A. S.

Publications and source records attributed to Grossman, A. S..

8 recordsLinked to original sources

bioRxiv2026

A domesticated prophage enzyme links bacterial metabolism, episymbiosis and phage fitness

Prophages are increasingly recognized as drivers of bacterial adaptation, yet how domesticated prophage genes shape microbial symbiosis remains poorly understood. Here, we identify GDPDXhp1, a glycerophosphodiester phosphodiesterase encoded by prophage Xhp1 of Schaalia odontolytica XH001, as a metabolic regulator linking bacterial physiology, episymbiosis and phage fitness. Association with the epibiotic bacterium Nanosynbacter lyticus TM7x strongly induced gdpdXhp1 expression and triggered GDPDXhp1-dependent lipid droplet accumulation in XH001. GDPDXhp1 orchestrated glycerophospholipid remodeling, membrane biophysical properties, respiration and cell-surface glycan composition in XH001, thereby promoting TM7x episymbiosis. Remarkably, GDPDXhp1 was also required for productive Xhp1 infection by remodeling susceptible bacterial host envelope structures to facilitate phage-host interactions. Together, our findings reveal a domesticated prophage enzyme that contributes to bacterial metabolism, interspecies symbiosis and viral reproduction, highlighting prophage as an overlooked source of metabolic innovation in microbial symbiosis.

microbiology↗

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

Resolving Host-Episymbiont Interaction Dynamics through Continuous Cultivation

Patescibacteria are an elusive linage of "microbial dark matter" bacteria predicted to represent [~]25% of total bacterial diversity. Despite this abundance and ubiquity, these organisms are challenging to cultivate, resulting from their specialized episymbiotic lifestyle. All cultivated representatives to date, predominantly composed of Saccharibacteria from the oral microbiome, depend on cognate prokaryotic hosts for growth and reproduction. Studying the growth dynamics of episymbiotic bacteria and their hosts in batch cultures has suggested that many episymbionts initially reduce host populations, and that hosts eventually adapt to episymbiont stress after serial passaging. However, discontinuous batch cultures do not reflect natural interactions between these organisms due to their drastically different growth rates. An episymbiont requires several ([~]2-4) serial passages alongside its host to reach the high cell densities needed to impact host growth, which complicates investigation of host inhibition and adaptation to episymbiont stress. To describe these dynamics accurately, we utilized continuous culture via small-scale Raspberry Pi powered bioreactors, called Pioreactors. Within a bioreactor, host bacteria can be cultivated at a consistent growth rate indefinitely, providing the perfect substrate for cultivation of model Saccharibacteria. Quantification of time until host crash, crash severity, time until recovery, and stable co-culture density provides mechanistic ways to describe episymbiont-host interactions. First, we used these techniques to compare episymbiont infection by three different episymbionts, revealing distinct infection patterns ranging from mild inhibition with rapid host adaptation, to rapid host collapse followed by "arms race" oscillation dynamics. Then, bioreactors were used to quantify the episymbiotic role played by a known host-binding type 4 pili (T4P-2), demonstrating that loss of long-distance host binding significantly delayed the host crash without altering general crash dynamics. These experiments reveal that episymbionts can have drastically different effects on bacterial communities and provide the tools necessary to describe strain/species differences and molecular interactions. ImportanceEpisymbiotic Patescibacteria represent one of the largest branches of life on Earth, as well as one of the least understood. Furthermore, because Patescibacteria can manipulate their hosts growth and morphology they have immense ecological potential to be shaping the communities they occupy, both environmental and microbiome-associated. Our study highlights for the first time the potential of small-scale continuous cultivation for studying episymbiotic interactions that cannot be captured in discontinuous cultures. Herein we used these techniques to interrogate inter-species variation in host inhibition potential and to determine how loss of a long-distance episymbiosis factor mechanistically alters the cycle of episymbiont infection; however, this cultivation platform will enable researchers to answer many new questions about these ubiquitous host-episymbiont interactions.

microbiology↗

bioRxiv2025

Episymbiotic Saccharibacteria suppresses epithelial immunoactivation through Type IV pili and TLR2 dependent endocytosis

Saccharibacteria are episymbionts that require host-bacteria to grow. They are positively associated with inflammatory diseases within the human microbiome, yet their mechanisms for interacting with the human host and contributing to diseases remain unknown. This study investigated interactions between a Saccharibacterium (Nanosynbacter lyticus), its host-bacteria (Schaalia odontolytica), and oral epithelial cells. The host-bacteria induced proinflammatory cytokines in epithelial cells, while Saccharibacteria were immune silent. Remarkably, Saccharibacteria dampened cytokine responses to host-bacteria during coinfection. This effect was driven by Saccharibacteria-induced clustering of TLR2 receptors, a process likely facilitated by type IV, ultimately leading to reduced TLR2-mediated cytokine signalling. High resolution imaging showed that Saccharibacteria were endocytosed by oral epithelial cells, and colocalized with endosome markers, eventually trafficking to lysosomes. Moreover, a subset of the Saccharibacteria survive endocytosis long-term, and retains their capability to reinfect host-bacteria, highlighting a mechanism for persistence in the oral microbiome and a vital role in mammalian immune system modulation.

immunology↗

bioRxiv2024

Saccharibacteria deploy two distinct Type IV pili, driving episymbiosis, host competition, and twitching motility

All cultivated Patescibacteria, or CPR, exist as obligate episymbionts on other microbes. Despite being ubiquitous in mammals and environmentally, molecular mechanisms of host identification and binding amongst ultrasmall bacterial episymbionts are largely unknown. Type 4 pili (T4P) are well conserved in this group and predicted to facilitate symbiotic interactions. To test this, we targeted T4P pilin genes in Saccharibacteria Nanosynbacter lyticus strain TM7x to assess their essentiality and roles in symbiosis. Our results revealed that N. lyticus assembles two distinct T4P, a non-essential thin pili that has the smallest diameter of any T4P and contributes to host-binding, episymbiont growth, and competitive fitness relative to other Saccharibacteria, and an essential thick pili whose functions include twitching motility. Identification of lectin-like minor pilins and modification of host cell walls suggest glycan binding mechanisms. Collectively our findings demonstrate that Saccharibacteria encode unique extracellular pili that are vital mediators of their underexplored episymbiotic lifestyle.

microbiology↗

bioRxiv2024

Bioinformatic discovery of type 11 secretion system (T11SS) cargo across the Proteobacteria

Type 11 secretion systems (T11SS) are broadly distributed among proteobacteria, with more than 3000 T11SS family outer membrane proteins (OMPs) comprising 10 major sequence similarity network (SSN) clusters. Of these, only 7, all from animal-associated cluster 1, have been experimentally verified as secretins of cargo, including adhesins, hemophores, and metal binding proteins. To identify novel cargo of a more diverse set of T11SS, we identified gene families co-occurring in gene neighborhoods with either cluster 1 or marine microbe-associated cluster 3 T11SS OMP genes. We developed bioinformatic controls to ensure perceived co-occurrences are specific to T11SS, and not general to OMPs. We found that both cluster 1 and cluster 3 T11SS OMPs frequently co-occur with single carbon metabolism and nucleotide synthesis pathways, but that only cluster 1 T11SS OMPs had significant co-occurrence with metal and heme pathways, as well as with mobile genetic islands, potentially indicating diversified function of this cluster. Cluster 1 T11SS co-occurrences included 2556 predicted cargo proteins, unified by the presence of a C-terminal {beta}-barrel domain, which fall into 141 predicted UniRef50 clusters and approximately 10 different architectures: 4 similar to known cargo and 6 uncharacterized types. We experimentally demonstrate T11SS-dependent secretion of an uncharacterized cargo type with homology to Plasmin sensitive protein (Pls). Unexpectedly, genes encoding marine cluster 3 T11SS OMPs only rarely co-occurred with the C-terminal {beta}-barrel domain and instead frequently co-occurred with DUF1194-containing genes. Overall, our results show that with sufficiently large-scale and controlled genomic data, T11SS-dependent cargo proteins can be accurately predicted. Impact StatementWe describe a novel method for controlling genomic neighborhood analyses and use this controlled co-occurrence technique to examine two distinct clusters of genes from the recently described type 11 secretion system (T11SS): Cluster 1, predominantly encoded by host-associated microbes and Cluster 3, encoded by marine microbes. We found that both clusters of predicted T11SS frequently co-occur with single carbon metabolism and nucleotide synthesis pathways, but only the host-associated cluster co-occurred with iron uptake pathways. Using these datasets, we predicted 2687 T11SS dependent cargo with approximately ten unique architectures, six of which have not previously been linked to T11SS. Finally, we validate our results by demonstrating T11SS-dependent secretion of a cargo protein with one of the novel architectures, plasmin-sensitive surface protein Pls from Haemophilus parahaemolyticus. Data SummaryThis paper describes genomic co-occurrences of T11SS OMP sequences originally identified through sequence similarity networking in a previous publication [3] as constituents of similarity clusters, 1 and 3. Accession IDs and assorted metadata for those clusters/networks are listed in Table S1 of that article: https://journals.asm.org/doi/suppl/10.1128/mbio.01956-21/suppl_file/mbio.01956-21-st001.xlsx. All other supporting data, accession IDs, code, and protocols have been provided within this article or through supplementary data files. Additionally, all supplemental data are available on Figshare (https://doi.org/10.6084/m9.figshare.28514069.v2) [1]. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/615229v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@c9123dorg.highwire.dtl.DTLVardef@cc7295org.highwire.dtl.DTLVardef@76a60corg.highwire.dtl.DTLVardef@18f6c6d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Bacterial hemophilin homologs and their specific type eleven secretor proteins have conserved roles in heme capture and are diversifying as a family

Cellular life relies on enzymes that require metal cofactors, which must be acquired from extracellular sources. Bacteria utilize surface and secreted proteins to acquire such valuable nutrients from their environment. These include the cargo proteins of the type eleven secretion system (T11SS), which have been connected to host specificity, metal homeostasis, and nutritional immunity evasion. This Sec-dependent, Gram-negative secretion system is encoded by organisms throughout the phylum Proteobacteria, including human pathogens Neisseria meningitidis, Proteus mirabilis, Acinetobacter baumannii, and Haemophilus influenzae. Experimentally verified T11SS-dependent cargo include host metal acquisition proteins transferrin binding protein B (TbpB) and lactoferrin binding protein B (LbpB), as well as the hemophilin homologs heme receptor protein C (HrpC) and hemophilin A (HphA), the complement immune evasion protein factor-H binding protein (fHbp), and the host symbiosis factor nematode intestinal localization protein C (NilC). Secretion of each of these cargo proteins relies on a specific T11SS. Here, we examined the specificity of T11SS systems for their cognate cargo proteins using taxonomically distributed homolog pairs of T11SS and hemophilin cargo and explore the ligand binding ability of those hemophilin homologs. Our comparative ligand binding analysis of four hemophilin family proteins identified previously unknown ligand binding diversity within this protein family, which informed our description of structural features that are likely to contribute to heme/porphyrin binding specificity. In vivo expression of hemophilin homologs revealed that each was secreted in a specific manner by its cognate T11SS protein. Furthermore, secretion assays of chimeric hemophilin proteins revealed that specificity is predominantly dictated by the C-terminal domain of the cognate cargo. Meanwhile, the N-terminal effector domains of these T11SS-dependent cargo proteins feature porphyrin binding pockets that drive ligand binding affinity and specificity. In light of these results, we have termed this N-terminal domain the hemophilin ligand binding domain (Hlb) after its first characterized representative.

microbiology↗

bioRxiv2020

Beyond Slam: the DUF560 family outer membrane protein superfamily SPAM has distinct network subclusters that suggest a role in non-lipidated substrate transport and bacterial environmental adaptation

In host-associated bacteria, surface and secreted proteins mediate acquisition of nutrients, interactions with host cells, and specificity of tissue-localization. In Gram-negative bacteria, the mechanism by which many proteins cross or become tethered to the outer membrane remains unclear. The domain of unknown function (DUF)560 occurs in outer membrane proteins throughout Proteobacteria and has been implicated in host-bacteria interactions and lipoprotein surface exposure. We used sequence similarity networking to reveal three subfamilies of DUF560 homologs. One subfamily includes those DUF560 proteins experimentally characterized to date: NilB, a host-range determinant of the nematode-mutualist Xenorhabdus nematophila, and the surface lipoprotein assembly modulators Slam1 and Slam2, which facilitate msurface exposure of lipoproteins in Neisseria meningitidis (1, 2). We show that DUF560 proteins from a second subfamily facilitate secretion of soluble, non-lipidated proteins across the outer membrane. Using in silico analysis, we demonstrate that DUF560 gene complement correlates with bacterial environment at a macro level and host association at a species level. The DUF560 protein superfamily represents a newly characterized Gram-negative secretion system capable of lipoprotein surface exposure and soluble protein secretion with conserved roles in facilitating symbiosis. In light of these data, we propose that it be titled the type eleven secretion system (TXISS). ImportanceThe microbial constituents of a host associated microbiome are decided by a complex interplay of microbial colonization factors, host surface conditions, and host immunological responses. Filling such niches requires bacteria to encode an arsenal of surface and secreted proteins to effectively interact with the host and co-occurring microbes. Bioinformatic predictions of the localization and function of putative bacterial colonization factors are essential for assessing the potential of bacteria to engage in pathogenic, mutualistic, or commensal activities. This study uses publicly available genome sequence data, alongside experimental results from representative gene products from Xenorhabdus nematophila, to demonstrate a role for DUF560 family proteins in the secretion of bacterial effectors of host interactions. Our research delineates a broadly distributed family of proteins and enables more accurate predictions of the localization of colonization factors throughout Proteobacteria.

microbiology↗