Biology subjects

Isberg, R. R.

Publications and source records attributed to Isberg, R. R..

27 records · Page 2Linked to original sources

bioRxiv2020

SdhA blocks disruption of the Legionella-containing vacuole by hijacking the OCRL phosphatase

Legionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion resulted in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA altered OCRL function, enlarging endosomes, driving endosomal accumulation of PI(4,5)P2, and interfering with endosomal trafficking. SdhA interrupted Rab GTPase-OCRL interactions by binding to the OCRL ASH domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulated OCRL and endosomal antigen EEA1, consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole.

microbiology↗

bioRxiv2020

Essential gene analysis in Acinetobacter baumannii by high-density transposon mutagenesis and CRISPR interference

Acinetobacter baumannii is a poorly understood bacterium capable of life-threatening infections in hospitals. Few antibiotics remain effective against this highly resistant pathogen. Developing rationally-designed antimicrobials that can target A. baumannii requires improved knowledge of the proteins that carry out essential processes allowing growth of the organism. Unfortunately, studying essential genes has been challenging using traditional techniques, which usually require time-consuming recombination-based genetic manipulations. Here, we performed saturating mutagenesis with dual transposon systems to identify essential genes in A. baumannii and we developed a CRISPR-interference (CRISPRi) system for facile analysis of these genes. We show that the CRISPRi system enables efficient transcriptional silencing in A. baumannii. Using these tools, we confirmed the essentiality of the novel cell division protein AdvA and discovered a previously uncharacterized AraC-family transcription factor (ACX60_RS03245) that is necessary for growth. In addition, we show that capsule biosynthesis is a conditionally essential process, with mutations in late-acting steps causing toxicity in strain ATCC 17978 that can be bypassed by blocking early-acting steps or activating the BfmRS stress response. These results open new avenues for analysis of essential pathways in A. baumannii. ImportanceNew approaches are urgently needed to control A. baumannii, one of the most drug resistant pathogens known. To facilitate the development of novel targets that allow inhibition of the pathogen, we performed a large-scale identification of genes whose products the bacterium needs for growth. We also developed a CRISPR-based gene knockdown tool that operates efficiently in A. baumannii, allowing rapid analysis of these essential genes. We used these methods to define multiple processes vital to the bacterium, including a previously uncharacterized gene-regulatory factor and export of a protective polymeric capsule. These tools will enhance our ability to investigate processes critical for the essential biology of this challenging hospital-acquired pathogen.

microbiology↗

bioRxiv2020

Topologically correct synthetic reconstruction of pathogen social behavior found in deep tissue sites

Within deep tissue sites, extracellular bacterial pathogens often replicate in clusters that are surrounded by immune cells. Disease is modulated by interbacterial interactions as well as bacterial-host cell interactions resulting in microbial growth, phagocytic attack and secretion of host antimicrobial factors. To overcome the limited ability to manipulate these infection sites, we established a system for Yersinia pseudotuberculosis (Yptb) growth in microfluidics-driven microdroplets that regenerates microbial social behavior in tissues. Chemical generation of nitric oxide (NO) in the absence of immune cells was sufficient to reconstruct microbial social behavior, as witnessed by expression of the NO-inactivating protein Hmp on the extreme periphery of microcolonies, mimicking spatial regulation in tissues. Similarly, activated macrophages that expressed inducible NO synthase (iNOS) drove peripheral expression of Hmp, allowing regeneration of social behavior observed in tissues. These results argue that topologically correct microbial tissue growth and associated social behavior can be reconstructed in culture.

microbiology↗

bioRxiv2020

Antibiotic hypersensitivity signatures identify targets for attack in the Acinetobacter baumannii cell envelope

Acinetobacter baumannii is an opportunistic pathogen that is a critical, high-priority target for new antibiotic development. Clearing of A. baumannii requires relatively high doses of antibiotics across the spectrum, primarily due to its protective cell envelope. Many of the proteins that support envelope integrity and modulate drug action are uncharacterized, largely because there is an absence of orthologs for several proteins that perform essential envelope-associated processes, impeding progress on this front. To identify targets that can synergize with current antibiotics, we performed an exhaustive analysis of A. baumannii mutants causing hypersensitivity to a multitude of antibiotic treatments. By examining mutants with antibiotic hypersensitivity profiles that parallel mutations in proteins of known function, we show that the function of poorly annotated proteins can be predicted and used to identify candidate missing link proteins in essential A. baumannii processes. Using this strategy, we uncovered multiple uncharacterized proteins with critical roles in cell division or cell elongation, and revealed that a predicted cell wall D,D-endopeptidase has an unappreciated function in lipooligosaccharide synthesis. Moreover, we provide a genetic strategy that uses hypersensitivity signatures to predict drug synergies, allowing the identification of {beta}-lactams that work cooperatively based on the cell wall assembly machineries that they preferentially target. These data reveal multiple pathways critical for envelope growth in A. baumannii that can be targeted in combination strategies for attacking the pathogen.

microbiology↗

bioRxiv2019

Entropy of a bacterial stress response is a generalizable predictor for fitness and antibiotic sensitivity.

Genes implicated in bacterial stress responses have been used to construct models that infer the growth outcome of a bacterium in the presence of antibiotics with the objective to develop novel diagnostic methods in the clinic. Current models are trained on data specific to a species or type of stress, making them potentially limited in their application. It is unclear if a generalizable response-signature exists that can predict bacterial fitness independent of strain, species or type of stress. Here we present a substantial RNA-Seq and experimental evolution dataset for 9 strains and species, under multiple antibiotic and non-antibiotic stress conditions. We show that gene panel-based models can accurately predict antibiotic mechanism of action, as well as the fitness outcome of Streptococcus pneumoniae in the presence of antibiotics or under nutrient depletion. However, these models quickly become species-specific as gene homology is limited. Instead, we define a new concept, transcriptomic entropy, which we use to quantify the amount of transcriptional disruption that occurs in a bacterium when responding to the environment. With entropy at the center, we train a suite of predictive (machine learning) models enabling generalizable fitness and antibiotic sensitivity predictions. These entropy-based models that predict bacterial fitness are validated for 7 Gram-positive and -negative species under antibiotic and non-antibiotic conditions indicating that transcriptomic entropy can be used as a generalizable stress signature. Moreover, rather than being a binary indicator of fitness, an entropy-based model was developed and validated to predict the minimum inhibitory concentration of an antibiotic. Lastly, we show that the inclusion of a varied-set of multi-omics features of a bacterial stress response further enhances fitness predictions by reducing ambiguity. By demonstrating the feasibility of generalizable predictions of bacterial fitness, this work establishes the fundamentals for potentially new approaches in infectious disease diagnostics, including antibiotic susceptibility testing.\n\nSignificance statementAccurate predictions of bacterial fitness outcome could potentially have clinical diagnostic value, such as predicting optimum antibiotic choice and dosage for treating infectious diseases. Existing models of fitness predictions rely mainly on gene panel approaches, which may be species- and stress-specific due to a lack of gene and response conservation. In order to overcome this limitation, we generated a substantial experimental dataset and identified entropy as a universal stress response signature that quantifies the level of transcriptional disruption that is indicative of fitness outcome under a stressful condition. We present and validate for Gram-positive and negative species a suite of entropy-based models that enable accurate predictions of fitness outcome and the level of antibiotic sensitivity in a species and stress-type independent manner.

systems biology↗

bioRxiv2019

Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole

Legionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonize vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the {Delta}sdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilize the replication vacuole, EEA1 and Rab11FIP1 showed enhanced colocalization with the vacuole surrounding the sdhA mutant compared with the WT vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant colocalization. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilize vacuole integrity during infection.

microbiology↗

bioRxiv2019

Iron-sulfur cluster repair contributes to Y. pseudotuberculosis survival within deep tissues

To successfully colonize host tissues, bacteria must respond to and detoxify many different host-derived antimicrobial compounds, such as nitric oxide (NO). NO has direct antimicrobial activity through attack on iron-sulfur (Fe-S) cluster-containing proteins. NO detoxification plays an important role in promoting bacterial survival, but it remains unclear if repair of Fe-S clusters is also important for bacterial survival within host tissues. Here we show that the Fe-S cluster repair protein, YtfE, contributes to the survival of Y. pseudotuberculosis within the spleen following nitrosative stress. Y. pseudotuberculosis forms clustered centers of replicating bacteria within deep tissues, where peripheral bacteria express the NO-detoxifying gene, hmp. ytfE expression also occurred specifically within peripheral cells at the edges of microcolonies. In the absence of ytfE, the area of microcolonies was significantly smaller than WT, consistent with ytfE contributing to the survival of peripheral cells. The loss of ytfE did not alter the ability of cells to detoxify NO, which occurred within peripheral cells in both WT and {Delta}ytfE microcolonies. In the absence of NO-detoxifying activity by hmp, NO diffused across {Delta}ytfE microcolonies, and there was a significant decrease in the area of microcolonies lacking ytfE, indicating that ytfE also contributes to bacterial survival in the absence of NO detoxification. These results indicate a role for Fe-S cluster repair in the survival of Y. pseudotuberculosis within the spleen, and suggest that extracellular bacteria may rely on this pathway for survival within host tissues.

microbiology↗

bioRxiv2018

Legionella pneumophila translocated translation inhibitors are required for bacterial-induced host cell cycle arrest

The cell cycle machinery controls diverse cellular pathways and is tightly regulated. Misregulation of cell division plays a central role in the pathogenesis of many disease processes. Various microbial pathogens interfere with the cell cycle machinery to promote host cell colonization. Although cell cycle modulation is a common theme among pathogens, the role that this interference plays in promoting diseases is unclear. Previously we demonstrated that the G1 and G2/M phases of the host cell cycle are permissive for Legionella pneumophila replication, while S phase provides a toxic environment for bacterial replication. In this study we show that L. pneumophila avoids host S phase by blocking host DNA synthesis and preventing cell cycle progression into S phase. Cell cycle arrest upon Legionella contact is dependent on the Icm/Dot secretion system. In particular, we found that cell cycle arrest is dependent on the intact enzymatic activity of translocated substrates that inhibits host translation. Moreover, we show that early in infection, the presence of these translation inhibitors is crucial to induce the degradation of the master regulator cyclin D1. Our results demonstrate that the bacterial effectors that inhibit translation are associated with preventing entry of host cells into a phase associated with restriction of L. pneumophila. Furthermore, control of cyclin D1 may be a common strategy used by intracellular pathogens to manipulate the host cell cycle and promote bacterial replication.\n\nSignificanceRecently, we showed that host cell cycle regulatory proteins control L. pneumophila growth. In particular, bacterial replication was found to be depressed in S-phase. This indicates that bacterial control of the host cell cycle can limit exposure of the pathogen to antimicrobial events that are cycle-specific. Here we uncovered bacterial factors that induce host cell cycle arrest by inhibiting host protein synthesis and preventing S phase transition. These data are consistent with S-phase toxicity serving as an important antimicrobial response that limits growth of some intracellular pathogens. Moreover, identification of microbial factors that block cell cycle progression and uncovering host cell cycle partners are candidates for future drug development. Our data point to a unifying role of the cell cycle in multiple disease processes.

microbiology↗

bioRxiv2018

The landscape of intrinsic and evolved fluoroquinolone resistance in Acinetobacter baumannii includes suppression of drug-induced prophage replication

The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii, the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (Topo IV). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug sensitive and resistant strains to identify loci that alter the sensitivity of the organism to ciprofloxacin. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectrum of mutations that altered drug sensitivity was found to be similar in the drug sensitive and double mutant gyrAparC background having resistance alleles in both genes. In contrast, introduction of a single gyrA resistance allele, resulting in preferential poisoning of Topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential Topo IV poisoning was induction of DNA synthesis in the region of two endogenous prophages, which appeared to occur in situ. Induction of the selective DNA synthesis in the gyrA background was also linked to enhanced activation of SOS response and heightened transcription of prophage genes relative to that observed in either the WT or gyrAparC double mutants. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to suppression of hyperactivation of the SOS response and endogenous prophage DNA synthesis.\n\nImportanceFluoroquinolones have been extremely successful antibiotics. Their clinical efficacy derives from the ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and Topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we sought to understand how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii, which causes resistant, life-threating infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to lower fluoroquinolone resistance independently of target alteration. Remarkably, fluoroquinolone targeting of Topo IV in specific mutants caused dramatic prophage hyperinduction, a response that was muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities.

microbiology↗