Browsing by Subject "Virulence Factors"

Now showing 1 - 8 of 8
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    Epigenetic regulation of the nitrosative stress response and intracellular macrophage survival by extraintestinal pathogenic Escherichia coli.
    (2011) Bateman, Stacey Lynn
    Escherichia coli is a typical constituent of the enteric tract in many animals, including humans. However, specialized extraintestinal pathogenic E. colistrains (ExPEC) may transition from benign occupation of the enteric and vaginal tracts to sterile sites such as the urinary tract, bloodstream, and central nervous system. ExPEC isolates of urinary tract origin express type 1 pili as a critical virulence determinant mediating adherence to and invasion into urinary tract tissues. Type 1 pili expression is under epigenetic regulation by a family of site-specific recombinases, including FimX, which is encoded from a genomic islet called PAI-X for Pathogenicity Islet of FimX. A goal of this study was to determine the prevalence of the type 1 pili epigenetic regulator genes (fimB, fimE, fimX, ipuA, ipuB) and associated PAI-X genes (hyxR, hyxA, hyxB) present among an extended, diverse collection of pathogenic and commensal E. coli isolates. Using a new multiplex PCR, fimX and the additional PAI-X genes were found to be highly associated with ExPEC (83.2%) and more prevalent in ExPEC of lower urinary tract origin (87.5%) than upper urinary tract origin (73.6%) or human commensal isolates (20.6%; p < 0.05, all comparisons). Fim-like recombinase genes ipuA and ipuB also had a significant association with ExPEC compared to commensal isolates, but had a low overall prevalence (23.8% vs. 11.1%; p < 0.05). PAI-X also showed a strong positive correlation with the presence of virulence genes in the genomes of pathogenic isolates. Combined, our molecular epidemiology studies indicate PAI-X is highly associated with ExPEC isolates, and its high prevalence suggests a potential role in the ExPEC lifestyle. Further investigation into the regulation of PAI-X factors showed that FimX is also an epigenetic regulator of a LuxR-like response regulator HyxR, encoded on PAI-X. In multiple clinical ExPEC isolates, FimX regulated hyxR expression through bidirectional phase inversion of its promoter region at sites different from the inversion sites of the type 1 pili promoter and independent of integration host factor IHF. Additional studies into the role of HyxR during ExPEC pathogenesis uncovered that HyxR is involved in regulation of the nitrosative stress response. In vitro, transition from high to low HyxR expression produced enhanced tolerance of reactive nitrogen intermediates (RNI), primarily through derepression of hmpA, encoding a nitric oxide detoxifying flavohemoglobin. However, in the macrophage, HyxR expression produced large effects on intracellular survival in the presence and absence of RNI, and independent of Hmp. Collectively, we have shown that the ability of ExPEC to survive in macrophages is contingent upon the proper transition from high to low HyxR expression through epigenetic regulatory control by FimX. ExPEC reside in the enteric tract as commensal reservoirs, but can transition to a pathogenic state by invading normally sterile niches, establishing infection, and disseminating to invasive sites like the bloodstream. Macrophages are required for ExPEC dissemination, suggesting the pathogen has developed mechanisms to persist within professional phagocytes. This study demonstrates the functional versatility of the FimX recombinase and identifies novel epigenetic and transcriptional regulatory controls for ExPEC tolerance to RNI challenge and survival during intracellular macrophage infection. Further investigation of these pathways may shed light on the regulatory cues and programming that provoke the commensal to pathogen transition.
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    Escherichia coli global gene expression in urine from women with urinary tract infection.
    (PLoS pathogens, 2010-11) Hagan, Erin C; Lloyd, Amanda L; Rasko, David A; Faerber, Gary J; Mobley, Harry LT
    Murine models of urinary tract infection (UTI) have provided substantial data identifying uropathogenic E. coli (UPEC) virulence factors and assessing their expression in vivo. However, it is unclear how gene expression in these animal models compares to UPEC gene expression during UTI in humans. To address this, we used a UPEC strain CFT073-specific microarray to measure global gene expression in eight E. coli isolates monitored directly from the urine of eight women presenting at a clinic with bacteriuria. The resulting gene expression profiles were compared to those of the same E. coli isolates cultured statically to exponential phase in pooled, sterilized human urine ex vivo. Known fitness factors, including iron acquisition and peptide transport systems, were highly expressed during human UTI and support a model in which UPEC replicates rapidly in vivo. While these findings were often consistent with previous data obtained from the murine UTI model, host-specific differences were observed. Most strikingly, expression of type 1 fimbrial genes, which are among the most highly expressed genes during murine experimental UTI and encode an essential virulence factor for this experimental model, was undetectable in six of the eight E. coli strains from women with UTI. Despite the lack of type 1 fimbrial expression in the urine samples, these E. coli isolates were generally capable of expressing type 1 fimbriae in vitro and highly upregulated fimA upon experimental murine infection. The findings presented here provide insight into the metabolic and pathogenic profile of UPEC in urine from women with UTI and represent the first transcriptome analysis for any pathogenic E. coli during a naturally occurring infection in humans.
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    Immunoinformatics approaches to explore Helicobacter Pylori proteome (Virulence Factors) to design B and T cell multi-epitope subunit vaccine.
    (Scientific reports, 2019-09) Khan, Mazhar; Khan, Shahzeb; Ali, Asim; Akbar, Hameed; Sayaf, Abrar Mohammad; Khan, Abbas; Wei, Dong-Qing
    Helicobacter Pylori is a known causal agent of gastric malignancies and peptic ulcers. The extremophile nature of this bacterium is protecting it from designing a potent drug against it. Therefore, the use of computational approaches to design antigenic, stable and safe vaccine against this pathogen could help to control the infections associated with it. Therefore, in this study, we used multiple immunoinformatics approaches along with other computational approaches to design a multi-epitopes subunit vaccine against H. Pylori. A total of 7 CTL and 12 HTL antigenic epitopes based on c-terminal cleavage and MHC binding scores were predicted from the four selected proteins (CagA, OipA, GroEL and cagA). The predicted epitopes were joined by AYY and GPGPG linkers. Β-defensins adjuvant was added to the N-terminus of the vaccine. For validation, immunogenicity, allergenicity and physiochemical analysis were conducted. The designed vaccine is likely antigenic in nature and produced robust and substantial interactions with Toll-like receptors (TLR-2, 4, 5, and 9). The vaccine developed was also subjected to an in silico cloning and immune response prediction model, which verified its efficiency of expression and the immune system provoking response. These analyses indicate that the suggested vaccine may produce particular immune responses against H. pylori, but laboratory validation is needed to verify the safety and immunogenicity status of the suggested vaccine design.
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    Influence of vancomycin minimum inhibitory concentration on the outcome of methicillin-susceptible Staphylococcus aureus left-sided infective endocarditis treated with antistaphylococcal β-lactam antibiotics: a prospective cohort study by the International Collaboration on Endocarditis.
    (Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases, 2017-08) Pericàs, JM; Messina, JA; Garcia-de-la-Mària, C; Park, L; Sharma-Kuinkel, BK; Marco, F; Wray, D; Kanafani, ZA; Carugati, M; Durante-Mangoni, E; Tattevin, P; Chu, VH; Moreno, A; Fowler, VG; Miró, JM; International Collaboration on Endocarditis Microbiology Investigators

    Objectives

    Left-sided methicillin-susceptible Staphylococcus aureus (MSSA) endocarditis treated with cloxacillin has a poorer prognosis when the vancomycin minimum inhibitory concentration (MIC) is ≥1.5 mg/L. We aimed to validate this using the International Collaboration on Endocarditis cohort and to analyse whether specific genetic characteristics were associated with a high vancomycin MIC (≥1.5 mg/L) phenotype.

    Methods

    All patients with left-sided MSSA infective endocarditis treated with antistaphylococcal β-lactam antibiotics between 2000 and 2006 with available isolates were included. Vancomycin MIC was determined by Etest as either high (≥1.5 mg/L) or low (<1.5 mg/L). Isolates underwent spa typing to infer clonal complexes and multiplex PCR for identifying virulence genes. Univariate analysis was performed to evaluate the association between in-hospital and 1-year mortality, and vancomycin MIC phenotype.

    Results

    Sixty-two cases met the inclusion criteria. Vancomycin MIC was low in 28 cases (45%) and high in 34 cases (55%). No significant differences in patient demographic data or characteristics of infection were observed between patients with infective endocarditis due to high and low vancomycin MIC isolates. Isolates with high and low vancomycin MIC had similar distributions of virulence genes and clonal lineages. In-hospital and 1-year mortality did not differ significantly between the two groups (32% (9/28) vs. 27% (9/34), p 0.780; and 43% (12/28) vs. 29% (10/34), p 0.298, for low and high vancomycin MIC respectively).

    Conclusions

    In this international cohort of patients with left-sided MSSA endocarditis treated with antistaphylococcal β-lactams, vancomycin MIC phenotype was not associated with patient demographics, clinical outcome or virulence gene repertoire.
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    Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.
    (MBio, 2015-09-29) Tenor, Jennifer L; Oehlers, Stefan H; Yang, Jialu L; Tobin, David M; Perfect, John R
    UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.
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    Structure of the Francisella response regulator QseB receiver domain, and characterization of QseB inhibition by antibiofilm 2-aminoimidazole-based compounds.
    (Molecular microbiology, 2017-10) Milton, Morgan E; Allen, C Leigh; Feldmann, Erik A; Bobay, Benjamin G; Jung, David K; Stephens, Matthew D; Melander, Roberta J; Theisen, Kelly E; Zeng, Daina; Thompson, Richele J; Melander, Christian; Cavanagh, John
    With antibiotic resistance increasing at alarming rates, targets for new antimicrobial therapies must be identified. A particularly promising target is the bacterial two-component system. Two-component systems allow bacteria to detect, evaluate and protect themselves against changes in the environment, such as exposure to antibiotics and also to trigger production of virulence factors. Drugs that target the response regulator portion of two-component systems represent a potent new approach so far unexploited. Here, we focus efforts on the highly virulent bacterium Francisella tularensis tularensis. Francisella contains only three response regulators, making it an ideal system to study. In this study, we initially present the structure of the N-terminal domain of QseB, the response regulator responsible for biofilm formation. Subsequently, using binding assays, computational docking and cellular studies, we show that QseB interacts with2-aminoimidazole based compounds that impede its function. This information will assist in tailoring compounds to act as adjuvants that will enhance the effect of antibiotics.
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    Structure-Guided Synthesis of FK506 and FK520 Analogs with Increased Selectivity Exhibit In Vivo Therapeutic Efficacy against Cryptococcus.
    (mBio, 2022-06) Hoy, Michael J; Park, Eunchong; Lee, Hyunji; Lim, Won Young; Cole, D Christopher; DeBouver, Nicholas D; Bobay, Benjamin G; Pierce, Phillip G; Fox, David; Ciofani, Maria; Juvvadi, Praveen R; Steinbach, William; Hong, Jiyong; Heitman, Joseph
    Calcineurin is an essential virulence factor that is conserved across human fungal pathogens, including Cryptococcus neoformans, Aspergillus fumigatus, and Candida albicans. Although an excellent target for antifungal drug development, the serine-threonine phosphatase activity of calcineurin is conserved in mammals, and inhibition of this activity results in immunosuppression. FK506 (tacrolimus) is a naturally produced macrocyclic compound that inhibits calcineurin by binding to the immunophilin FKBP12. Previously, our fungal calcineurin-FK506-FKBP12 structure-based approaches identified a nonconserved region of FKBP12 that can be exploited for fungus-specific targeting. These studies led to the design of an FK506 analog, APX879, modified at the C-22 position, which was less immunosuppressive yet maintained antifungal activity. We now report high-resolution protein crystal structures of fungal FKBP12 and a human truncated calcineurin-FKBP12 bound to a natural FK506 analog, FK520 (ascomycin). Based on information from these structures and the success of APX879, we synthesized and screened a novel panel of C-22-modified compounds derived from both FK506 and FK520. One compound, JH-FK-05, demonstrates broad-spectrum antifungal activity in vitro and is nonimmunosuppressive in vivo. In murine models of pulmonary and disseminated C. neoformans infection, JH-FK-05 treatment significantly reduced fungal burden and extended animal survival alone and in combination with fluconazole. Furthermore, molecular dynamic simulations performed with JH-FK-05 binding to fungal and human FKBP12 identified additional residues outside the C-22 and C-21 positions that could be modified to generate novel FK506 analogs with improved antifungal activity. IMPORTANCE Due to rising rates of antifungal drug resistance and a limited armamentarium of antifungal treatments, there is a paramount need for novel antifungal drugs to treat systemic fungal infections. Calcineurin has been established as an essential and conserved virulence factor in several fungi, making it an attractive antifungal target. However, due to the immunosuppressive action of calcineurin inhibitors, they have not been successfully utilized clinically for antifungal treatment in humans. Recent availability of crystal structures of fungal calcineurin-bound inhibitor complexes has enabled the structure-guided design of FK506 analogs and led to a breakthrough in the development of a compound with increased fungal specificity. The development of a calcineurin inhibitor with reduced immunosuppressive activity and maintained therapeutic antifungal activity would add a significant tool to the treatment options for these invasive fungal infections with exceedingly high rates of mortality.
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    Variation in chromosome copy number influences the virulence of Cryptococcus neoformans and occurs in isolates from AIDS patients.
    (BMC Genomics, 2011-10-27) Hu, Guanggan; Wang, Joyce; Choi, Jaehyuk; Jung, Won Hee; Liu, Iris; Litvintseva, Anastasia P; Bicanic, Tihana; Aurora, Rajeev; Mitchell, Thomas G; Perfect, John R; Kronstad, James W
    BACKGROUND: The adaptation of pathogenic fungi to the host environment via large-scale genomic changes is a poorly characterized phenomenon. Cryptococcus neoformans is the leading cause of fungal meningoencephalitis in HIV/AIDS patients, and we recently discovered clinical strains of the fungus that are disomic for chromosome 13. Here, we examined the genome plasticity and phenotypes of monosomic and disomic strains, and compared their virulence in a mouse model of cryptococcosis RESULTS: In an initial set of strains, melanin production was correlated with monosomy at chromosome 13, and disomic variants were less melanized and attenuated for virulence in mice. After growth in culture or passage through mice, subsequent strains were identified that varied in melanin formation and exhibited copy number changes for other chromosomes. The correlation between melanin and disomy at chromosome 13 was observed for some but not all strains. A survey of environmental and clinical isolates maintained in culture revealed few occurrences of disomic chromosomes. However, an examination of isolates that were freshly collected from the cerebrospinal fluid of AIDS patients and minimally cultured provided evidence for infections with multiple strains and copy number variation. CONCLUSIONS: Overall, these results suggest that the genome of C. neoformans exhibits a greater degree of plasticity than previously appreciated. Furthermore, the expression of an essential virulence factor and the severity of disease are associated with genome variation. The occurrence of chromosomal variation in isolates from AIDS patients, combined with the observed influence of disomy on virulence, indicates that genome plasticity may have clinical relevance.