Browsing by Author "Brothwell, Julie A."
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Item Advances and Obstacles in the Genetic Dissection of Chlamydial Virulence(Springer, 2018) Brothwell, Julie A.; Muramatsu, Matthew K.; Zhong, Guangming; Nelson, David E.; Microbiology and Immunology, School of MedicineObligate intracellular pathogens in the family Chlamydiaceae infect taxonomically diverse eukaryotes ranging from amoebae to mammals. However, many fundamental aspects of chlamydial cell biology and pathogenesis remain poorly understood. Genetic dissection of chlamydial biology has historically been hampered by a lack of genetic tools. Exploitation of the ability of chlamydia to recombine genomic material by lateral gene transfer (LGT) ushered in a new era in chlamydia research. With methods to map mutations in place, genetic screens were able to assign functions and phenotypes to specific chlamydial genes. Development of an approach for stable transformation of chlamydia also provided a mechanism for gene delivery and platforms for disrupting chromosomal genes. Here, we explore how these and other tools have been used to test hypotheses concerning the functions of known chlamydial virulence factors and discover the functions of completely uncharacterized genes. Refinement and extension of the existing genetic tools to additional Chlamydia spp. will substantially advance understanding of the biology and pathogenesis of this important group of pathogens.Item Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation(American Society for Microbiology, 2016-09-19) Muramatsu, Matthew K.; Brothwell, Julie A.; Steinman, Barry D.; Putman, Timothy E.; Rockey, Daniel D.; Nelson, David E.; Department of Microbiology & Immunology, IU School of MedicineChlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells.Item CpxA Phosphatase Inhibitor Activates CpxRA and Is a Potential Treatment for Uropathogenic Escherichia coli in a Murine Model of Infection(American Society for Microbiology, 2022-04-27) Fortney, Kate R.; Smith, Sara N.; van Rensburg, Julia J.; Brothwell, Julie A.; Gardner, Jessi J.; Katz, Barry P.; Ahsan, Nagib; Duerfeldt, Adam S.; Mobley, Harry L.T.; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineCpxRA is an envelope stress response system that is highly conserved in the Enterobacteriaceae. CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR (CpxR-P), a transcription factor. In response to membrane stress, CpxR-P is produced and upregulates genes involved in membrane repair and downregulates genes that encode virulence factors that are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and in uropathogenic Escherichia coli (UPEC) are attenuated in murine models. We hypothesized that pharmacologic activation of CpxR could serve as an antimicrobial/antivirulence strategy and recently showed that 2,3,4,9-tetrahydro-1H-carbazol-1-amines activate the CpxRA system by inhibiting CpxA phosphatase activity. Here, we tested the ability of a series of three CpxRA-activating compounds with increasing potency to clear UPEC stain CFT073 in a murine urinary tract infection model. We show that these compounds are well tolerated and achieve sufficient levels to activate CpxR in the kidneys, bladder, and urine. Although the first two compounds were ineffective in promoting clearance of CFT073 in the murine model, the most potent derivative, compound 26, significantly reduced bacterial recovery in the urine and trended toward reducing bacterial recovery in the bladder and kidneys, with efficacy similar to ciprofloxacin. Treatment of CFT073 cultured in human urine with compound 26 fostered accumulation of CpxR-P and decreased the expression of proteins involved in siderophore biosynthesis and binding, heme degradation, and flagellar movement. These studies suggest that chemical activation of CpxRA may present a viable strategy for treating infections due to UPEC. IMPORTANCE: The increasing prevalence of urinary tract infections (UTIs) due to antibiotic-resistant uropathogenic Escherichia coli (UPEC) is a major public health concern. Bacteria contain proteins that sense their environment and have no human homologs and, thus, are attractive drug targets. CpxRA is a conserved sensing system whose function is to reduce stress in the bacterial cell membrane; activation of CpxRA reduces the expression of virulence determinants, which must cross the cell membrane to reach the bacterial surface. We previously identified a class of compounds that activate CpxRA. We show in a mouse UTI model that our most potent compound significantly reduced recovery of UPEC in the urine, trended toward reducing bacterial recovery in the bladder and kidneys, did not kill UPEC, and downregulated multiple proteins involved in UPEC virulence. Since these compounds do not act by a killing mechanism, they have potential to treat UTIs caused by antibiotic-resistant bacteria.Item Dispensability of Ascorbic Acid Uptake and Utilization Encoded by ulaABCD for the Virulence of Haemophilus ducreyi in Humans(Oxford University Press, 2023) Brothwell, Julie A.; Fortney, Kate R.; Batteiger, Teresa; Katz, Barry P.; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineCompared with wounded skin, ascorbic acid is enriched in pustules of humans experimentally infected with Haemophilus ducreyi. Compared with the broth-grown inocula, transcription of the H. ducreyi ulaABCD operon, which encodes genes for ascorbic acid uptake, is increased in pustules. We hypothesized that ascorbic acid uptake plays a role in H. ducreyi virulence. Five volunteers were infected with both H. ducreyi strain 35000HP and its isogenic ulaABCD deletion mutant at multiple sites; the papule and pustule formation rates of the mutant and parent strains were similar. Thus, ascorbic acid uptake is not essential for H. ducreyi virulence in humans.Item Formate production is dispensable for Haemophilus ducreyi virulence in human volunteers(American Society for Microbiology, 2023) Brothwell, Julie A.; Fortney, Kate R.; Williams, Jalan S.; Batteiger, Teresa A.; Duplantier, Rory; Grounds, Danielle; Jannasch, Amber S.; Katz, Barry P.; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineHaemophilus ducreyi is a causative agent of cutaneous ulcers in children who live in the tropics and of the genital ulcer disease chancroid in sexually active persons. In the anaerobic environment of abscesses and ulcers, anaerobic respiration and mixed acid fermentation (MAF) can be used to provide cellular energy. In Escherichia coli, MAF produces formate, acetate, lactate, succinate, and ethanol; however, MAF has not been studied in H. ducreyi. In human challenge experiments with H. ducreyi 35000HP, transcripts of the formate transporter FocA and pyruvate formate lyase (PflB) were upregulated in pustules compared to the inocula. We made single and double mutants of focA and pflB in 35000HP. Growth of 35000HPΔfocA was similar to 35000HP, but 35000HPΔpflB and 35000HPΔfocA-pflB had growth defects during both aerobic and anaerobic growth. Mutants lacking pflB did not secrete formate into the media. However, formate was secreted into the media by 35000HPΔfocA, indicating that H. ducreyi has alternative formate transporters. The pH of the media during anaerobic growth decreased for 35000HP and 35000HPΔfocA, but not for 35000HPΔpflB or 35000HPΔfocA-pflB, indicating that pflB is the main contributor to media acidification during anaerobic growth. We tested whether formate production and transport were required for virulence in seven human volunteers in a mutant versus parent trial between 35000HPΔfocA-pflB and 35000HP. The pustule formation rate was similar for 35000HP (42.9%)- and 35000HPΔfocA-pflB (62%)-inoculated sites. Although formate production occurs during in vitro growth and focA-pflB transcripts are upregulated during human infection, focA and pflB are not required for virulence in humans.Item Genes Differentially Expressed by Haemophilus ducreyi during Anaerobic Growth Significantly Overlap Those Differentially Expressed during Experimental Infection of Human Volunteers(American Society for Microbiology, 2022) Brothwell, Julie A.; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineHaemophilus ducreyi causes cutaneous ulcers in children and the genital ulcer disease chancroid in adults. In humans, H. ducreyi is found in the anaerobic environment of an abscess; previous studies comparing bacterial gene expression levels in pustules with the inocula (∼4-h aerobic mid-log-phase cultures) identified several upregulated differentially expressed genes (DEGs) that are associated with anaerobic metabolism. To determine how H. ducreyi alters its gene expression in response to anaerobiosis, we performed RNA sequencing (RNA-seq) on both aerobic and anaerobic broth cultures harvested after 4, 8, and 18 h of growth. Principal-coordinate analysis (PCoA) plots showed that anaerobic growth resulted in distinct transcriptional profiles compared to aerobic growth. During anaerobic growth, early-time-point comparisons (4 versus 8 h) identified few DEGs at a 2-fold change in expression and a false discovery rate (FDR) of <0.01. By 18 h, we observed 18 upregulated and 16 downregulated DEGs. DEGs involved in purine metabolism, the uptake and use of alternative carbon sources, toxin production, nitrate reduction, glycine metabolism, and tetrahydrofolate synthesis were upregulated; DEGs involved in electron transport, thiamine biosynthesis, DNA recombination, peptidoglycan synthesis, and riboflavin synthesis or modification were downregulated. To examine whether transcriptional changes that occur during anaerobiosis overlap those that occur during infection of human volunteers, we compared the overlap of DEGs obtained from 4 h of aerobic growth to 18 h of anaerobic growth to those found between the inocula and pustules in previous studies; the DEGs significantly overlapped. Thus, a major component of H. ducreyi gene regulation in vivo involves adaptation to anaerobiosis. IMPORTANCE: In humans, H. ducreyi resides in the anaerobic environment of an abscess and appears to upregulate genes involved in anaerobic metabolism. How anaerobiosis alone affects gene transcription in H. ducreyi is unknown. Using RNA-seq, we investigated how anaerobiosis affects gene transcription over time compared to aerobic growth. Our results suggest that a substantial component of H. ducreyi gene regulation in vivo overlaps the organism's response to anaerobiosis in vitro. Our data identify potential therapeutic targets that could be inhibited during in vivo growth.Item Genetic Screen in Chlamydia muridarum Reveals Role for an Interferon-Induced Host Cell Death Program in Antimicrobial Inclusion Rupture(American Society for Microbiology, 2019-04-09) Giebel, Amanda M.; Hu, Shuai; Rajaram, Krithika; Finethy, Ryan; Toh, Evelyn; Brothwell, Julie A.; Morrison, Sandra G.; Suchland, Robert J.; Stein, Barry D.; Coers, Jörn; Morrison, Richard P.; Nelson, David E.; Microbiology and Immunology, School of MedicineInterferon-regulated immune defenses protect mammals from pathogenically diverse obligate intracellular bacterial pathogens of the genus Chlamydia Interferon gamma (IFN-γ) is especially important in controlling the virulence of Chlamydia species and thus impacts the modeling of human chlamydial infection and disease in mice. How IFN-γ contributes to cell-autonomous defenses against Chlamydia species and how these pathogens evade IFN-γ-mediated immunity in their natural hosts are not well understood. We conducted a genetic screen which identified 31 IFN-γ-sensitive (Igs) mutants of the mouse model pathogen Chlamydia muridarum Genetic suppressor analysis and lateral gene transfer were used to map the phenotype of one of these mutants, Igs4, to a missense mutation in a putative chlamydial inclusion membrane protein, TC0574. We observed the lytic destruction of Igs4-occupied inclusions and accompanying host cell death in response to IFN-γ priming or various proapoptotic stimuli. However, Igs4 was insensitive to IFN-γ-regulated cell-autonomous defenses previously implicated in anti-Chlamydia trachomatis host defense in mice. Igs4 inclusion integrity was restored by caspase inhibitors, indicating that the IFN-γ-mediated destruction of Igs4 inclusions is dependent upon the function of caspases or related prodeath cysteine proteases. We further demonstrated that the Igs4 mutant is immune restricted in an IFN-γ-dependent manner in a mouse infection model, thereby implicating IFN-γ-mediated inclusion destruction and host cell death as potent in vivo host defense mechanisms to which wild-type C. muridarum is resistant. Overall, our results suggest that C. muridarum evolved resistance mechanisms to counter IFN-γ-elicited programmed cell death and the associated destruction of intravacuolar pathogens.IMPORTANCE Multiple obligatory intracellular bacteria in the genus Chlamydia are important pathogens. In humans, strains of C. trachomatis cause trachoma, chlamydia, and lymphogranuloma venereum. These diseases are all associated with extended courses of infection and reinfection that likely reflect the ability of chlamydiae to evade various aspects of host immune responses. Interferon-stimulated genes, driven in part by the cytokine interferon gamma, restrict the host range of various Chlamydia species, but how these pathogens evade interferon-stimulated genes in their definitive host is poorly understood. Various Chlamydia species can inhibit death of their host cells and may have evolved this strategy to evade prodeath signals elicited by host immune responses. We present evidence that chlamydia-induced programmed cell death resistance evolved to counter interferon- and immune-mediated killing of Chlamydia-infected cells.Item Genome Copy Number Regulates Inclusion Expansion, Septation, and Infectious Developmental Form Conversion in Chlamydia trachomatis(American Society for Microbiology, 2021-01-11) Brothwell, Julie A.; Brockett, Mary; Banerjee, Arkaprabha; Stein, Barry D.; Nelson, David E.; Liechti, George W.; Microbiology and Immunology, School of MedicineDNA replication is essential for the growth and development of Chlamydia trachomatis, however it is unclear how this process contributes to and is controlled by the pathogen's biphasic lifecycle. While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature sensitive dnaE mutant (dnaEts ) that exhibits a ∼100-fold reduction in genome copy number at the non-permissive temperature (40°C), but replicates similarly to the parent at the permissive temperature of 37°C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in dnaEts at 40°C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. dnaEts formed fewer and smaller pathogenic vacuoles (inclusions) at 40°C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernable peptidoglycan and polymerized MreB, the major cell division organizing protein in Chlamydia responsible for nascent peptidoglycan biosynthesis. We also found that absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion reversed when dnaEts was shifted from 40°C to 37°C early in infection, and intragenic suppressor mutations in dnaE also restored dnaEts genome replication and inclusion expansion at 40°C. Overall, our results show that genome replication in C. trachomatis is required for inclusion expansion, septum formation, and the transition between the microbe's replicative and infectious forms.SIGNIFICANCE Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and non-infectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen's infectious cycle has not been characterized. We show that genome replication is dispensable for EB to RB conversion, but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in Chlamydia that facilitate the ordering of events in the microbe's biphasic life cycle. Our findings suggest that Chlamydia utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment.Item Haemophilus ducreyi Infection Induces Oxidative Stress, Central Metabolic Changes, and a Mixed Pro- and Anti-inflammatory Environment in the Human Host(American Society for Microbiology, 2022) Brothwell, Julie A.; Fortney, Kate R.; Gao, Hongyu; Wilson, Landon S.; Andrews, Caroline F.; Tran, Tuan M.; Hu, Xin; Batteiger, Teresa A.; Barnes, Stephen; Liu, Yunlong; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineFew studies have investigated host-bacterial interactions at sites of infection in humans using transcriptomics and metabolomics. Haemophilus ducreyi causes cutaneous ulcers in children and the genital ulcer disease chancroid in adults. We developed a human challenge model in which healthy adult volunteers are infected with H. ducreyi on the upper arm until they develop pustules. Here, we characterized host-pathogen interactions in pustules using transcriptomics and metabolomics and examined interactions between the host transcriptome and metabolome using integrated omics. In a previous pilot study, we determined the human and H. ducreyi transcriptomes and the metabolome of pustule and wounded sites of 4 volunteers (B. Griesenauer, T. M. Tran, K. R. Fortney, D. M. Janowicz, et al., mBio 10:e01193-19, 2019, https://doi.org/10.1128/mBio.01193-19). While we could form provisional transcriptional networks between the host and H. ducreyi, the study was underpowered to integrate the metabolome with the host transcriptome. To better define and integrate the transcriptomes and metabolome, we used samples from both the pilot study (n = 4) and new volunteers (n = 8) to identify 5,495 human differentially expressed genes (DEGs), 123 H. ducreyi DEGs, 205 differentially abundant positive ions, and 198 differentially abundant negative ions. We identified 42 positively correlated and 29 negatively correlated human-H. ducreyi transcriptome clusters. In addition, we defined human transcriptome-metabolome networks consisting of 9 total clusters, which highlighted changes in fatty acid metabolism and mitigation of oxidative damage. Taken together, the data suggest a mixed pro- and anti-inflammatory environment and rewired central metabolism in the host that provides a hostile, nutrient-limited environment for H. ducreyi.Item Interactions of the Skin Pathogen Haemophilus ducreyi With the Human Host(Frontiers Media, 2021-02-03) Brothwell, Julie A.; Griesenauer, Brad; Chen, Li; Spinola, Stanley M.; Microbiology and Immunology, School of MedicineThe obligate human pathogen Haemophilus ducreyi causes both cutaneous ulcers in children and sexually transmitted genital ulcers (chancroid) in adults. Pathogenesis is dependent on avoiding phagocytosis and exploiting the suppurative granuloma-like niche, which contains a myriad of innate immune cells and memory T cells. Despite this immune infiltrate, long-lived immune protection does not develop against repeated H. ducreyi infections—even with the same strain. Most of what we know about infectious skin diseases comes from naturally occurring infections and/or animal models; however, for H. ducreyi, this information comes from an experimental model of infection in human volunteers that was developed nearly three decades ago. The model mirrors the progression of natural disease and serves as a valuable tool to determine the composition of the immune cell infiltrate early in disease and to identify host and bacterial factors that are required for the establishment of infection and disease progression. Most recently, holistic investigation of the experimentally infected skin microenvironment using multiple “omics” techniques has revealed that non-canonical bacterial virulence factors, such as genes involved in central metabolism, may be relevant to disease progression. Thus, the immune system not only defends the host against H. ducreyi, but also dictates the nutrient availability for the invading bacteria, which must adapt their gene expression to exploit the inflammatory metabolic niche. These findings have broadened our view of the host-pathogen interaction network from considering only classical, effector-based virulence paradigms to include adaptations to the metabolic environment. How both host and bacterial factors interact to determine infection outcome is a current focus in the field. Here, we review what we have learned from experimental H. ducreyi infection about host-pathogen interactions, make comparisons to what is known for other skin pathogens, and discuss how novel technologies will deepen our understanding of this infection.