Biology subjects

Kirby, J. E.

Publications and source records attributed to Kirby, J. E..

12 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↗

bioRxiv2025

Poloxamer dilution as an on-demand alternative to agar dilution-based antimicrobial susceptibility testing

Agar dilution is a reference susceptibility testing method uniquely or preferentially recommended for certain antimicrobials. However, the effort required to pour individual agar plates spanning a doubling dilution range precludes its practical implementation in hospital clinical laboratories. Here, we describe an on-demand replacement for agar dilution (AD), specifically substituting poloxamer 407 (also known as Pluronic F-127) for Bacto Agar as the solidifying agent. Notably, 20% poloxamer 407 solutions (e.g., with Mueller-Hinton broth) remain liquid at refrigerated temperatures, but solidify upon warming, enabling facile set-up of poloxamer dilution testing (PD) in Petri dish or microwell format. For fosfomycin susceptibility testing, PD and reference agar dilution (AD) showed excellent categorical (CA) and essential (EA) agreement for E. coli (100% and 87%, respectively, n=31). For other Enterobacterales, excluding Klebsiella spp; CA and EA were both 82% (n=17, respectively). For P. aeruginosa, CA and EA were 60% and 100% (n=10), respectively, with the lower CA reflecting the large number of strains tested with MICs near categorical breakpoints. There were no very major errors, while major errors were only observed for Klebsiella spp. Additionally, PD substantially reduced the number of skipped dilutions 6-fold for E. coli (P < 0.0001) and inhibited swarming of Proteus species. We conclude that PD and AD, an imperfect gold standard, have essentially equivalent practical performance and that PD can therefore serve as an on-demand alternative testing methodology in clinical laboratories for fosfomycin testing of gram-negative pathogens. A broader exploration of PDs utility is thus warranted. ImportanceAccurate antibiotic susceptibility testing is essential for guiding treatment of bacterial infections. For the antibiotic fosfomycin, used to treat Escherichia coli urinary tract infections, the most reliable testing method requires solid media prepared by hand for each antibiotic concentration, which is too time consuming for most clinical laboratories to perform. Our study shows that replacing agar with an alternative temperature-sensitive gelling agent called poloxamer enables laboratories to prepare solid test plates rapidly without special equipment. This approach, which is essentially identical to traditional agar dilution, provides a practical alternative that can be implemented by hospital clinical microbiology laboratories for accurate fosfomycin testing near the point of patient care. This strategy may also be applicable to other drugs for which agar dilution is the preferred testing method, supporting expedited testing to inform treatment decisions for bacterial infections.

microbiology↗

bioRxiv2025

Enhanced Activity of Apramycin and Apramycin-Based Combinations Against Mycobacteroides abscessus

BackgroundMycobacteroides abscessus are rapidly growing non-tuberculous mycobacteria that cause chronic lung and soft tissue infections. Treatment options are often severely limited due to intrinsic resistance to most antimicrobials. Amikacin has historically been a mainstay of combination treatment regimens. However, irreversible hearing loss and vestibular toxicity have led to a search for alternative agents. Apramycin is a novel aminoglycoside currently in phase I clinical trials that may offer lower potential for ototoxic and renal toxic side effects. ObjectivesThe goal of this study was to compare apramycins in vitro activity with amikacin and other aminoglycosides against a large collection of M. abscessus clinical isolates, both alone and in combination with clofazimine or linezolid. We also tested the activity of apramycin against a more limited collection of other species of rapidly growing mycobacteria. MethodsAnalysis was performed using reference broth microdilution minimal inhibitory concentration testing, inkjet printer-assisted checkerboard assays, and time-kill assays. ResultsAgainst M. abscessus, the MIC50/90 for apramycin (2 {micro}g/mL) was 8-fold lower than for amikacin (16 {micro}g/mL). Plazomicin was inactive, and organisms were rarely susceptible to tobramycin. Synergy was not detected by checkerboard assay. In time-kill studies, clofazimine modestly potentiated activity of apramycin and. to a lesser extent, amikacin. Apramycin and amikacin showed delayed bacterial killing that either achieved or approached a bactericidal threshold. Apramycin was similarly potent against other rapidly growing mycobacteria tested. ConclusionsApramycin exhibits more potent in vitro activity against a diverse set of M. abscessus and other rapidly growing mycobacteria than approved aminoglycosides.

microbiology↗

bioRxiv2025

Triple antimicrobial combinations with potent synergistic activity against M. abscessus

Synergy of antimicrobial combinations was tested against contemporary Mycobacteroides abscessus isolates using 2-D and 3-D checkerboard assays. Triple combinations of omadacycline-azithromycin-clofazimine, tigecycline-azithromycin-clofazimine, omadacycline-azithromycin-linezolid and omadacycline-azithromycin-contezolid demonstrated synergy (FIC [≤] 0.5) against 33%, 31%, 62%, and 66% of isolates, respectively. Notably, in all triple combinations, macrolide resistant M. abscessus subsp. abscessus and M. abscessus subsp. bolletii isolates were fully sensitized to azithromycin at the FIC index, as were isolates with elevated clofazimine MICs.

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

Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance

BackgroundCarbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms. MethodsTo overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem susceptible Enterobacterales, isolated from patients from four U.S. hospitals over nearly five years. ResultsOur CRE collection comprised a diverse range of species, lineages and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment. ConclusionsCollectively, the framework we developed provides a clearer, high resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

genomics↗

bioRxiv2021

Transcriptomics reveals how minocycline-colistin synergy overcomes antibiotic resistance in multidrug-resistant Klebsiella pneumoniae

Multidrug resistant gram-negative bacteria are a rapidly growing public health threat, and the development of novel antimicrobials has failed to keep pace with their emergence. Synergistic combinations of individually ineffective drugs present a potential solution, yet little is understood about the mechanisms of most such combinations. Here, we show that the combination of colistin (polymyxin E) and minocycline has a high rate of synergy against colistin-resistant and minocycline-intermediate or -resistant strains of Klebsiella pneumoniae. Furthermore, using RNA-Seq, we characterized the transcriptional profiles of these strains when treated with the drugs individually and in combination. We found a striking similarity between the transcriptional profiles of bacteria treated with the combination of colistin and minocycline at individually subinhibitory concentrations and those of the same isolates treated with minocycline alone. We observed a similar pattern with the combination of polymyxin B nonapeptide (a polymyxin B analogue that lacks intrinsic antimicrobial activity) and minocycline. We also found that genes involved in polymyxin resistance and peptidoglycan biosynthesis showed significant differential gene expression in the different treatment conditions, suggesting possible mechanisms for the antibacterial activity observed in the combination. These findings suggest that the synergistic activity of this combination against bacteria resistant to each drug alone involves sublethal outer membrane disruption by colistin, which permits increased intracellular accumulation of minocycline. IMPORTANCEFew treatment options exist for multidrug resistant gram-negative bacteria. Synergistic antimicrobial combinations (in which two individually inactive drugs regain activity when used in combination) can overcome resistance. In susceptible bacteria, minocycline acts inside the cell by preventing protein synthesis, while colistin disrupts the outer membrane. Even in bacteria resistant to both drugs, the combination of the two is active. To understand this activity, we studied Klebsiella pneumoniae isolates resistant to both drugs. We extracted messenger RNA during treatment with each drug alone and with the combination, then performed RNA sequencing to analyze gene expression levels. Bacteria treated with the combination had an expression profile similar to bacteria treated with minocycline alone. Our findings suggest that synergy results from low-level, subinhibitory membrane disruption by colistin, which permits sufficient accumulation of minocycline in the cell to overcome resistance. Understanding this pathway will facilitate further development of synergistic antimicrobial combination regimens.

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↗

bioRxiv2021

Amotosalen is a bacterial multidrug efflux pump substrate potentially affecting its pathogen inactivation activity

Pathogen inactivation is a strategy to improve the safety of transfusion products. The Cerus Intercept technology makes use of a psoralen compound called amotosalen in combination with UVA light to inactivate bacteria, viruses and protozoa. Psoralens have structural similarity to bacterial multidrug-efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens and with recent reported outbreaks of transfusion-associated sepsis with Acinetobacter, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through microbiological, biophysical and molecular modeling analysis. The main efflux systems in Enterobacterales and Acinetobacter baumannii, tripartite RND (resistance-nodulation-cell division) systems which span the inner and outer membranes of Gram-negative pathogens and expel antibiotics from the bacterial cytoplasm into the extracellular space, were specifically examined. We found that amotosalen was an efflux substrate for the TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump from Acinetobacter baumannii, and that minimal inhibitory concentrations for contemporary bacterial isolates in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens. ImportancePathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by the major TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump in Acinetobacter baumannii. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the minimal inhibitory concentrations for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

microbiology↗

bioRxiv2021

Synergistic Activity of Minocycline and Rifampin in Combination with Antifungal Drugs against Candida auris

Candida auris is an emerging multidrug-resistant fungal pathogen that spreads readily in healthcare settings and has caused numerous hospital outbreaks. Very few treatment options exist for C. auris infections. We evaluated the activity of all two-drug combinations of three antifungal agents (amphotericin B, caspofungin, and voriconazole) and two antibacterial agents (minocycline and rifampin) against a collection of 10 C. auris isolates using an automated, inkjet printer-assisted checkerboard array method. Three antibacterial-antifungal combinations (amphotericin B plus rifampin, amphotericin B plus minocycline, and caspofungin plus minocycline) demonstrated synergistic activity by checkerboard array against [≥]90% of strains. The two amphotericin B-containing combinations were also synergistic using the time-kill synergy testing method. Our results suggest that combinations of antifungal and antibacterial agents may provide a promising avenue for treatment of this multidrug-resistant pathogen.

microbiology↗

bioRxiv2020

SARS-CoV2 Testing: The Limit of Detection Matters

Resolving the COVID-19 pandemic requires diagnostic testing to determine which individuals are infected and which are not. The current gold standard is to perform RT-PCR on nasopharyngeal samples. Best-in-class assays demonstrate a limit of detection (LoD) of ~100 copies of viral RNA per milliliter of transport media. However, LoDs of currently approved assays vary over 10,000-fold. Assays with higher LoDs will miss more infected patients, resulting in more false negatives. However, the false-negative rate for a given LoD remains unknown. Here we address this question using over 27,500 test results for patients from across our healthcare network tested using the Abbott RealTime SARS-CoV-2 EUA. These results suggest that each 10-fold increase in LoD is expected to increase the false negative rate by 13%, missing an additional one in eight infected patients. The highest LoDs on the market will miss a majority of infected patients, with false negative rates as high as 70%. These results suggest that choice of assay has meaningful clinical and epidemiological consequences. The limit of detection matters.

microbiology↗