Biology subjects

Kang, Y.-S.

Publications and source records attributed to Kang, Y.-S..

4 recordsLinked to original sources

bioRxiv2026

Genome-wide identification of metabolic and regulatory determinants of intracellular growth in Brucella neotomae

We used transposon sequencing (Tn-seq) to define genetic requirements for intracellular survival of Brucella neotomae, a rodent-associated species. A near-saturating mutant library was subjected to selection during infection of J774A.1 macrophages, identifying 54 genes required for intracellular fitness. These included core components of the VirB type IV secretion system, multiple regulatory factors, an aquaporin gene with a strong fitness defect, and a set of metabolic genes involved in amino acid biosynthesis. Targeted mutagenesis revealed that methionine and histidine biosynthesis are indispensable for intracellular growth, whereas tryptophan biosynthesis was required for full intracellular fitness, with mutants exhibiting significant but incomplete attenuation. Notably, these auxotrophs grew normally in minimal medium under axenic conditions, indicating that their requirement is specific to the intracellular environment. Amino acid supplementation rescued intracellular growth in a concentration- and time-dependent manner, consistent with increased metabolic demand during intracellular replication. Disruption of the aquaporin gene similarly impaired intracellular survival, suggesting a role for water homeostasis during adaptation to the macrophage vacuolar environment. Beyond metabolic and osmotic adaptation, we identify OmpR1 as an upstream regulator of B. neotomae virulence. Biochemical, genetic, and transcriptional analyses establish a hierarchical regulatory cascade in which OmpR1 activates the BvrR/BvrS system, which in turn controls VjbR and downstream VirB expression. Under the conditions examined, OmpR1 is required for activation of this cascade. Consistent with this, OmpR1 loss is not rescued by VjbR and requires BvrR activity for restoration of intracellular growth. Phylogenetic analysis places OmpR1 in a distinct lineage relative to other well-characterized Brucella transcriptional regulators, suggesting that this regulatory pathway has been underappreciated across the genus. Together, these findings reveal that intracellular fitness in Brucella depends on metabolic capacity, osmotic homeostasis, and a hierarchical regulatory cascade centered on OmpR1. Author SummaryBrucella species are bacteria that survive and replicate inside immune cells called macrophages, where they cause persistent infection. To live within these cells, the bacteria must carefully balance their metabolism with the expression of genes required for virulence. We used a genome-wide genetic approach to determine which genes are specifically required for intracellular survival of Brucella neotomae, a rodent-associated species. We found that several amino acid biosynthesis pathways, including those required to produce methionine and histidine, are essential for replication inside macrophages but are not required during growth in laboratory media. This indicates that the intracellular environment imposes nutrient limitations not apparent in culture. We also discovered that a gene encoding an aquaporin, which regulates water movement across the bacterial membrane, is critical for intracellular survival, highlighting the importance of maintaining water balance within the host cell vacuole. In addition, we identify OmpR1 as an upstream regulator that controls a hierarchical virulence cascade required for intracellular growth. Our findings show that successful infection depends on metabolic capacity, virulence regulation and water homeostasis, and provide new insight into how Brucella adapts to its host environment.

microbiology↗

bioRxiv2026

Host GPCR-cAMP signaling balances Gαs and Gαi activity to control intracellular Brucella infection

In this study, we investigated the impact of G protein-coupled receptor (GPCR) signaling on the intracellular replication of the model pathogen Brucella neotomae. Building on a prior chemical genetics screen, we identified agonists of the Gi-coupled adenosine A1 and dopamine D4 receptors as potent inhibitors of intracellular Brucella replication. In contrast, agonists of Gs-coupled adenosine A2A or dopamine D1 receptors, as well as antagonists of A1 or D4 receptors, either failed to inhibit or enhanced intracellular replication. Wild-type B. neotomae induced a rapid, type IV secretion system-dependent increase in host-cell cAMP during early infection. ENBA and cilostamide prevented this infection-associated cAMP increase and completely inhibited intracellular growth; this effect was partially reversed by cell-permeable cAMP analogs Using a real-time NanoBRET biosensor, we detected rapid Gs activation within minutes of infection that was sustained during wild-type but not {Delta}virB4 infection and was abrogated by ENBA or cilostamide. Disruption of early Gs-cAMP signaling redirected BCVs to replication-incompatible phagolysosomal and autophagy-associated compartments. Collectively, these data support a model in which early GPCR signaling dynamics, balancing Gs and Gi pathways, are critical for establishment of productive intracellular Brucella infection. Importance StatementBrucella species cause chronic infections by surviving and multiplying inside immune cells. To do this, Brucella must remodel the membrane-bound compartment that surrounds it after uptake, steering it away from destructive lysosomes and toward a permissive niche where replication can occur. We found that Brucella rapidly triggers a host signaling response controlled by G protein-coupled receptors, leading to a rise in a common cellular messenger molecule (cAMP) within minutes of infection. This early signal depends on the bacterial type IV secretion system and is required to build the replication-permissive compartment. When we disrupted this signaling with small molecules, bacteria were rerouted into degradative, autophagy-associated compartments and failed to establish productive infection. These results reveal an early host signaling checkpoint that Brucella exploits to create its intracellular niche and suggest that targeting host signaling dynamics, rather than bacterial viability directly, may provide new strategies to block intracellular infection.

microbiology↗

bioRxiv2022

A Versatile Nanoluciferase Reporter Reveals Structural Properties Associated With a Highly Efficient, N-Terminal Legionella pneumophila type IV Secretion Translocation Signal

Many gram-negative pathogens rely on type IV secretion systems (T4SS) for infection. One limitation in the field has been the lack of ideal reporters to identify T4SS translocated effectors and study T4SS function. Most existing reporter systems make use of fusions to reporter proteins, for example, {beta}-lactamase, to detect translocated enzymatic activity inside the host cell. However, these systems require costly substrates, complex procedures to separate eukaryotic cytoplasm for analysis, and/or are insensitive. Here, we developed and characterized a novel reporter system using nanoluciferase (NLuc) fusions to address these limitations. Serendipitously, we discovered that Nluc itself is efficiently translocated by L. pneumophila T4SS in an IcmSW chaperone-dependent manner via an N-terminal translocation signal. Extensive directed and random mutagenesis in the NLuc N-terminus revealed a critical -helical domain spanning D5 to V9, as mutations that are predicted to disrupt this -helix were translocation defective. Notably, NLuc was capable of translocating several proteins examined when fused to the N or C-terminus, while maintaining robust luciferase activity. In particular, it delivered the split GFP11 fragment into J774 macrophages permanently transfected with GFPopt, thereby resulting in in vivo assembly of superfolder GFP. This provided a bifunctional assay in which translocation could be assayed in by fluorescence microplate, confocal microscopy, and/or luciferase assay. We further identified an optimal NLuc substrate, which allowed a robust, inexpensive, one-step, high throughput screening assay to identify T4SS translocation substrates and inhibitors. Taken, together NLuc provides both new insight into and tools for studying T4SS biology. ImportanceType IV secretion systems (T4SS) are used by gram-negative pathogens to coopt host cell function. However, the translocation signals recognized by T4SS are not fully explained by primary amino acid sequence, suggesting yet to be defined contributions of secondary and tertiary structure. Here, we unexpectedly identify nanoluciferase (NLuc) as an efficient IcmSW-dependent translocated T4SS substrate and provide extensive mutagenesis data suggesting that the first N-terminal, alpha helix domain is a critical translocation recognition motif. Notably, most existing reporter systems for studying translocated proteins make use of fusions to reporters to permit detection of translocated enzymatic activity inside the host cell. However, existing systems require extremely costly substrates, complex technical procedures to isolate eukaryotic cytoplasm for analysis, and/or are insensitive. Importantly, we find that NLuc provides a powerful, cost-effective new tool to address these limitations and facilitate high throughput exploration of secretion system biology.

microbiology↗

bioRxiv2021

Profiling the in vitro and in vivo activity of streptothricin-F against carbapenem-resistant Enterobacterales: a historic scaffold with a novel mechanism of action

Streptothricins are components of the natural product, nourseothricin; each containing identical streptolidine and gulosamine aminosugar moieties attached to varying numbers of linked {beta}-lysines. Nourseothricin was discovered by Waksman and colleagues in the early 1940s, generating intense interest because of excellent Gram-negative activity. However, the natural product mixture was associated with toxicity, and subsequent exploration was limited. Here, we establish the activity spectrum of nourseothricin and its main components, streptothricin-F (S-F, one lysine) and streptothricin D (S-D, three lysines), purified to homogeneity, against highly drug-resistant, carbapenem-resistant Enterobacterales (CRE). The MIC50 and MIC90 for S-F and S-D were 2 and 4 {micro}M, and 0.25 and 0.5 {micro}M, respectively. S-F and nourseothricin showed rapid, bactericidal activity. S-F and S-D both showed approximately 40-fold greater selectivity for prokaryotic than eukaryotic ribosomes in in vitro translation assays. There was >10-fold in vitro selectivity of S-F compared with S-D on LLC-PK1 and J774 cell lines. In vivo, delayed renal toxicity occurred at >10-fold higher doses of S-F compared with S-D. Substantial treatment effect of S-F in the murine thigh model was observed against the otherwise pandrug-resistant, NDM-1-expressing Klebsiella pneumoniae Nevada strain at dosing levels without observable or minimal toxicity. Resistance mutations obtained in single ribosomal operon E. coli identify novel interactions with 16S rRNA helix 34, i.e., C1504A and A1196G/C conferred high level resistance to nourseothricin. Based on promising, unique activity, we suggest that the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, gram-negative pathogens. IMPORTANCEStreptothricins are a historic class of antibiotics discovered by Waksman and colleagues in the 1940s. Toxicities associated with the streptothricin natural product mixture, also known as nourseothricin, discouraged further development. However, we found that a component of nourseothricin, streptothricin-F, retained potent activity against contemporary carbapenem-resistant Enterobacterales with significant selectivity in in vitro and in vivo assays. This included demonstration of rapid bactericidal activity in vitro and substantial therapeutic effect in the murine thigh model against a pandrug-resistant Klebsiella pneumoniae isolate at non-toxic concentrations. Through resistance mutation analysis, we identified helix 34 of 16S rRNA in the prokaryotic ribosome, and specifically bases C1054 and A1196, as critical for streptothricins activity. The mechanism of action is distinct from other known translation inhibitors. Based on promising and unique activity, we believe the streptothricin scaffold deserves further pre-clinical exploration as a potential therapeutic for the treatment of CRE and potentially other multidrug-resistant, Gram-negative pathogens.

microbiology↗